Labeling of immobilized proteins using dipyrrometheneboron difluoride dyes

ABSTRACT

The invention describes methods for labeling or detecting of immobilized poly(amino acids), including peptides, polypeptides and proteins, on membranes and other solid supports, using fluorescent dipyrrometheneboron difluoride dyes. Such immobilized poly(amino acids) are labeled or detected on blots or on arrays of poly(amino acids), or are attached to immobilized aptamers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/210,438 filedAug. 23, 2005, which is a division of U.S. Ser. No. 10/005,050, filedDec. 3, 2001, which disclosures are herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to labeling or detecting of poly(amino acids),including peptides, polypeptides and proteins, that are immobilized onmembranes and other solid supports, using dipyrrometheneboron difluoridedyes.

BACKGROUND OF THE INVENTION

Poly(amino acids) are typically immobilized on membranes and other solidsupports, as in blots or arrays, to enable analysis of the poly(aminoacids). Labeling or detection of such immobilized poly(amino acids) isof great importance in a multitude of diverse activities, ranging frombasic research to enzyme production, forensics analysis and diagnostics.When analyzing the immobilized poly(amino acids), ideally the totalpoly(amino acid) profile is labeled in a way that will not interferewith other subsequent analytical methods that may be directed atspecific targets within the profile.

When analyzing complex mixtures of proteins and other poly(amino acids),e.g., mixtures extracted from cells, cell organelles, tissues, or otherbiological samples, the first step involves some method of separatingsuch complex mixtures into discrete locations or bands, typically by 1-Dor 2-D gel electrophoresis. Proteins in a given band are oftenidentified by using a specific binding pair member, such as an antibodyor lectin, that binds selectively to the proteins in that band. However,in order to be accessible to antibodies or other specific binding pairmembers, the protein bands must first be transferred from the gel to asolid support (electroblotted), such as a membrane. In some cases, thecomplex mixtures are generated in the form of arrays that are alreadyimmobilized on a solid support, and the specific binding pair member isused to probe the array of immobilized proteins or other poly(aminoacids) for those having certain defined characteristics. In addition tothe more traditional arrays of proteins, the discovery of the SELEX®(Systematic Evolution of Ligands by EXponential enrichment) processenabled the identification of nucleic acid-based ligands, referred to asaptamers, that recognize molecules other than nucleic acids, includingproteins, with high affinity and specificity (Ann. Rev. Biochem. 64, 763(1995); Chem. Rev. 97, 349 (1997) and U.S. Pat. No. 5,270,163). Ingeneral, arrays of aptamers are bound to a solid support, and after amixture of proteins or other poly(amino acids) is applied to the aptamerarray, unbound poly(amino acids) are removed. The poly(amino acids) thatare immobilized by binding to the aptamer array can be counterstainedand probed.

In all cases, the specific binding pair members that are used toidentify proteins in a band or location of interest can also be used toascertain a number of important characteristics of the protein,including the presence and quantity of the protein in the band, themolecular weight of the protein, and the efficiency of proteinextraction or separation. Such specific binding pair members, however,are only useful for the analysis of the proteins that are selectivelybound by the specific binding pair member and do not yield anyinformation about the proteins in other bands. Therefore, a counterstainis typically used to detect all the other proteins in the mixture toprovide a frame of reference for the analysis.

Counterstains for proteins like Coomassie Brilliant Blue (CBB), AmidoBlack, silver nitrate (silver staining), and colloidal gold are oftenused as calorimetric counterstains for electroblotted proteins (Anal.Biochem. 164, 303 (1987)). Although most of these methods have been incommon use for many years, they suffer from certain limitations. CBBstaining is easy to perform and shows good reproducibility but is notvery sensitive and only allows the detection of the major componentswithin a protein sample (typically, not more than 200-300 spots on ablot from a cell extract that may actually contain thousands ofindividual proteins). In addition, the calorimetric counterstains suchas Amido Black and CBB bind so tightly to proteins that destaining isdifficult and residual counterstain often blocks epitopes for subsequentimmunodetection (Meth. Mol. Biol. Ch. 35, 313 (1999); Anal. Biochem.276, 129 (1999); Anal. Biochem. 202, 100 (1992)). Silver staining is upto 100× more sensitive than CBB staining, which makes it suitable forthe detection of trace components within a protein sample (the detectionlimit is ˜0.1 ng protein), and is still a method of choice foranalytical gels. The major drawbacks to silver staining are the poorreproducibility, the limited dynamic range, and the fact that certainproteins stain poorly, negatively or not at all. Silver staining is alsoa procedure that is labor intensive and requires carefully timed stepsto achieve reproducibility. Membranes stained with colloidal gold havealso been found to generate heavy backgrounds after applyingchemiluminescence-based immunodetection methods (Electrophoresis 21,2196 (2000)), and staining lightly enough to yield useful resultsresulted in poor reproducibility as the endpoint had to be determinedsubjectively by the practitioner. Typically, all of thesecounterstaining procedures require that replicate blots be run, onestained with a calorimetric detection technique and the other blotprobed for specific proteins using immunological methods. However, inorder to identify proteins, the two blots have to be aligned. This leadsto problems because often two blots from the same sample may differ insize due to swelling or shrinking during the transfer and stainingprocedures and precise alignment of the replicate blot is difficult(Biotechniques 30, 266, (2001)).

In contrast, fluorescent counterstains have certain common advantagesover the colored stains, although the performance characteristics ofcertain classes of fluorescent counterstains differ substantially.Generally, the fluorescent counterstains that have been describedprovide a greater linear dynamic range for quantitation and, are easierto visualize on CCD camera-based and laser scanner-based imagingsystems. Moreover, they do not interfere with photographic documentationof colored reaction products generated by enzyme-catalyzed detectionsystems in common use, as the fluorescent counterstains are not visibleupon white light illumination, except with particularly abundantproteins. Fluorescent counterstaining of the total protein profiles onnitrocellulose or PVDF membranes has been described using, e.g.,2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF), SYPRO Rose Plus dye, SYPRORuby dye, fluorescamine, fluorescein isothiocyanate (FITC),dichlorotriazinylaminofluorescein (DTAF), and dansyl chloride(Electrophoresis 21, 1123 (2000); BioTechniques 28, 944 (2000)). Amethod for creating a permanent protein record using MDPF has been used.However, MDPF is not compatible with laser-based gel scanners since itdoes not absorb significantly in the visible region of the spectrum andits absorbance in the ultraviolet is also less than that of thepreferred visible light-excitable dyes of this invention(Electrophoresis 21, 1123 (2000); BioTechniques 28, 944 (2000)). Inaddition, MDPF-labeled blots must be viewed using wet PVDF membranes, asfluorescence signal decreases 500-fold upon drying (Electrophoresis 19,2407 (1998)). The fluorescent metal chelate dye, SYPRO Ruby protein blotstain, has been successfully incorporated into immunodetectionprocedures employing calorimetric, fluorescent or chemiluminescentdetection reagents (Anal. Biochem. 276, 129 (1999); Electrophoresis 22,881 (2001); Electrophoresis 21, 2196, 2208 (2000)). However, staining ofimmobilized proteins by SYPRO Rose Plus and SYPRO Ruby protein blotstains (both of which only bind noncovalently to proteins) is easilyreversible and both dyes are washed away during the blocking step priorto incubation with antibodies. Thus, they do not provide a permanentrecord of total protein on the blot as do the dyes of this invention.Reactive fluorescent counterstains such as fluorescamine, FITC, DTAF anddansyl chloride have also been used to create a permanent fluorescentprotein record (Anal. Biochem. 164, 303 (1987); J. Biochem. Biophys.Methods 46, 31 (2000)). However, these dyes are not very photostable andtheir fluorescence tends to be environment (dansyl) and/or pH(fluoresceins) sensitive. In addition, the linear range of fluorescenceis limited by fluorescence quenching at high concentrations (see Table 3in Example 28). The current invention allows for a permanent record ofboth total protein and specific protein(s) with high sensitivity on asingle blot and yields significantly greater sensitivity than that ofthe previously described fluorescent dyes for total protein staining,including the fluorescein-based dyes (see Table 3 in Example 28). Blotscan be viewed using standard UV illumination, by illumination with axenon-arc source or with a dual-wavelength laser-based gel scanner. Inparticular, all of the preferred dyes of the present invention haveabsorption maxima at >495 nm and extinction coefficients of >50,000cm⁻¹M⁻¹. Thus, they can be excited by the argon-ion laser, bylonger-wavelength lasers and laser diodes and by other commonlong-wavelength excitation sources that are typically utilized in theequipment that yields the most sensitive detection of proteins.

The current invention has several distinct advantages over knownmethods. The current invention uses dipyrrometheneboron difluoride dyes(e.g., various substituted 4-bora-3a,4a-diaza-s-indacene difluoride dyessold by Molecular Probes, Inc. under the trademark BODIPY) as acounterstain for immobilized poly(amino acids). These dyes have beenfound to have a variety of advantageous properties, including highextinction coefficients, a high fluorescence quantum yield, spectra thatare essentially insensitive to solvent polarity and pH, a narrowemission bandwidth, resulting in a higher peak intensity than other dyessuch as fluorescein, a greater photostability than fluorescein in someenvironments and lack of an ionic charge (U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; U.S. Pat. Nos. 5,274,113; 5,451,663; and MOLECULARPROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (“MPHANDBOOK”) by Richard P. Haugland, 6^(th) Ed., (1996), and itssubsequent 7^(th) edition and 8^(th) edition updates issued on CD Rom inNovember 1999 and May 2001, respectively, each of which are incorporatedby reference). However, it is known that dipyrrometheneboron difluoridedyes, including BODIPY dyes, usually show significant fluorescencequenching when attached to proteins (see, e.g., U.S. Pat. No. 5,719,031;MP HANDBOOK, supra, 6^(th) edition at p. 14; and Anal Biochem 251, 144(1997)), particularly when proteins are labeled with a large molarexcess of the reactive dipyrrometheneboron difluoride dye. Unexpectedly,it was found that certain dipyrrometheneboron difluoride dyes that areused to label proteins or other poly(amino acids) immobilized on a solidsurface do not suffer from this reported quenching phenomenon.Unexpectedly, this severe quenching was not observed in the presentinvention when the proteins were detected following blotting onto amembrane, even when the proteins were reacted with a vast excess of thereactive dye. In addition, in contrast to colorimetric counterstainingmethods, the current invention permits simultaneous dichromaticvisualization of a target protein and the remaining proteins in theprofile on a single electroblot. Since the selected dipyrrometheneborondifluoride counterstains also absorb significantly in the UV region ofthe spectrum, blots can be visualized using a handheld UV lamp or aUV-epi-illumination system in conjunction with a photographic or CCDcamera. These counterstains also absorb in the visible region of thespectrum, and they may be detected with a dual-wavelength laser-basedgel scanner using a photomultiplier tube. Unlike some other reactivedyes that have been used to detect proteins on a solid support, theblots can be imaged either wet or dry, as the selecteddipyrrometheneboron difluoride counterstains do not fade upon drying.Furthermore, the use of dipyrrometheneboron difluoride counterstainsdoes not interfere with the subsequent methods commonly used to analyzeimmobilized proteins.

Thus, there is an unmet need for an easy to use and sensitive method forthe labeling and detection (and optionally quantitation) of totalproteins and other poly(amino acids) on membranes and other solidsupports, that would overcome the disadvantages and limitations ofcurrent methods. Such methods would be useful in research, forensics,quality control and medical diagnostics. This invention meets these andother needs.

SUMMARY OF THE INVENTION

Poly(amino acids) that are immobilized on a solid support are labeledaccording to the present invention by incubating the poly(amino acids)with a labeling mixture that comprises one or more chemically reactivedipyrrometheneboron difluoride dyes for a time sufficient for the dye toform a covalent bond with the poly(amino acids), and then removing anyunbound dye by washing the immobilized poly(amino acids) with a suitablesolvent. Typically, the solid support is made of solvent-resistantmaterials such as nylon, poly(vinylidene difluoride) (PVDF), glass,plastic, and their derivatives. The immobilized poly(amino acids)generally have a molecular weight between 500 and 200,000 daltons. Forbest results, the dye is present in the labeling mixture in aconcentration of 0.01 micromolar to 10 micromolar. Subsequent additionof a specific binding pair member that binds selectively to a target ortargets within the sample of immobilized poly(amino acids) is apreferred aspect of the invention.

In one aspect of the invention, the poly(amino acids) are separated bygel electrophoresis prior to being transferred to a solid support forlabeling with the dipyrrometheneboron difluoride dyes. In another aspectof the invention, the poly(amino acids) are immobilized in arrays on thesolid support for labeling with the dipyrrometheneboron difluoride dyes.In yet another aspect of the invention, the poly(amino acids) areimmobilized by being selectively bound to their specific aptamersarrayed on a solid support

After the immobilized poly(amino acids) are labeled with thedipyrrometheneboron difluoride dyes, whether blotted from a gel orformatted in arrays, the unbound dye is removed (typically by washing,e.g., with a solvent) and the labeled poly(amino acids) are detected byilluminating the bound dipyrrometheneboron difluoride dyes to yield adetectable optical response, typically a fluorescent optical response,and using the detectable optical response to detect the correspondingpoly(amino acids). Dipyrrometheneboron difluoride dyes having anexcitation peak between 495 nm and 640 nm are particularly useful forthe invention. In one aspect of the invention, following reaction withthe immobilized proteins and washing to remove unconjugated dyes, theprotein conjugates are illuminated for 5 seconds or less.

In one aspect of the invention, the proteins or other poly(amino acids)are bound to aptamers that are, in turn, previously arrayed on a solidsupport. Aptamers can be used as a diagnostic or prognostic tool. In oneembodiment, arrays of aptamers are bound to a solid support, and aprotein sample is applied. Unbound protein is washed off, anddipyrrometheneboron difluoride dyes that covalently label reactive sitesin proteins but not nucleic acids are used to generate protein profiles.The labeled proteins on aptamers are detected by using an array reader,which uses the same light sources as are commonly used in commerciallyavailable blot readers.

In aspects of the invention that include adding a specific binding pairmember that binds selectively to a target or targets within theimmobilized poly(amino acids), the specific binding pair member istypically a lectin, a biotin-binding protein, an antibody or an antibodyfragment. In one aspect of the invention, a label is covalently attachedto the specific binding pair member. Alternatively, the specific bindingpair member is unlabeled, but a secondary binding pair member is added,to which secondary binding pair member a label is covalently attachedand which secondary binding pair member binds selectively to thespecific binding pair member (e.g., the secondary binding pair member isa secondary antibody and the specific binding pair member is a primaryantibody). The label on the secondary binding pair member or specificbinding pair member is typically a fluorescent dye, a biotin, a haptensuch as digoxigenin, or an enzyme (e.g., a peroxidase, luciferase,aequorin, glycosidase or phosphatase). Where the label is an enzyme, afluorogenic or chemiluminescent substrate (e.g., a fluorescent tyramide,a polyfluorinated xanthene, a fluorogenic quinazolinone, DDAO phosphate,luciferin, coelenterazine or a dioxetane phosphate) is typically addedfor detection of the target. Where the target of the specific bindingpair member is a biotinylated protein, a biotin-binding protein (e.g.,an avidin) to which a label is covalently attached is used for detectionof the target. Where the label is a hapten that is not biotin, anantibody to that hapten is utilized in the detection scheme, forinstance if the hapten is digoxigenin the specific binding pair membercomprises an antibody to digoxigenin. Unbound specific binding pairmembers or secondary binding pair members are removed for best detectionresults.

In one further aspect of the invention, the specific binding pair memberis a chemical stain such as a thiol-reactive dye that recognizes onlythose proteins that contain thiols, a dye that selectively reacts withoxidized glycoproteins such as Pro-Q Emerald 300 (U.S. patent Ser. No.09/970,215) or Pro-Q Sapphire 365 or Pro-Q Sapphire 488 oligohistidinestains (Molecular Probes, Inc).

In a further embodiment of the invention, kits adapted for the practiceof any of the claimed methods are described. Such kits typically includeone or more dipyrrometheneboron difluoride dyes and one or more specificbinding pair members or secondary binding pair members to which a labelis covalently attached. Where the label is an enzyme that is capable ofutilizing a fluorogenic, chromogenic or chemiluminescent substrate, theappropriate fluorogenic, chromogenic or chemiluminescent substrate isincluded.

The methods of the invention are broadly applicable to labeling anypoly(amino acid) from any origin. The methods are especially useful andapplicable to labeling poly(amino acids) from biological material. Suchpoly(amino acids) may be contained in cells or cellular materials andwill typically be obtained for the purposes of immunoassaying todetermining the presence or location of a specific target, diagnosingmedical conditions, or identifying disease states. These and otheraspects of the invention are described in more detail below.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In the present invention, a novel method of labeling and/or detectingimmobilized poly(amino acids) is disclosed, and novel kits forpracticing such methods are provided. To facilitate understanding of theinvention, the disclosure of the invention is organized in sections, asfollows. First, a definition section is provided to define terms andphrases used commonly throughout the disclosure. This definition sectionincludes a comprehensive description of the types of immobilizedpoly(amino acids), solid supports, and labeled specific binding pairmembers that can be used with the invention. The next section describesspecific dipyrrometheneboron difluoride dyes used in labeling thepoly(amino acids) in methods of the invention. The following sectiondescribes methods of the invention for labeling or detecting immobilizedpoly(amino acids). This next section is a description of the kitsadapted for practicing the methods of the invention. Then variousapplications and expansions of the invention are described; thisdescription is followed by detailed examples illustrating the invention.

DEFINITIONS

To assist in the understanding of the invention, the following terms, asused herein, are defined below.“Aptamer” means a nucleic acid that binds selectively to an intendedtarget molecule (“target,” as defined below). This binding interactiondoes not encompass standard nucleic acid/nucleic acid hydrogen bondformation exemplified by Watson-Crick base pair formation (e.g., A bindsto U or T and G bind to C), but encompasses all other types ofnon-covalent (or in some cases covalent) binding. Non-limiting examplesof non-covalent binding include hydrogen bond formation, electrostaticinteraction, Van der Waals interaction and hydrophobic interaction. Anaptamer may bind to another molecule by any or all of these types ofinteraction, or in some cases by covalent interaction. Covalent bindingof an aptamer to another molecule may occur where the aptamer or targetmolecule contains a chemically reactive or photoreactive moiety. Theterm “aptamer” or “specifically binding nucleic acid” refers to anucleic acid that is capable of forming a complex with an intendedtarget substance. “Target-specific” means that the aptamer binds to atarget analyte with a much higher affinity than it binds tocontaminating materials.“Array” means a two-dimensional spatial grouping or an arrangement ofbiomolecules.“Binding pair” refers to first and second molecules that bindselectively to each other. A specific binding pair member is the firstmember of the binding pair, and binds selectively to the second memberof the binding pair, which is its complement or complementary bindingpair member. The binding between the members of a binding pair istypically noncovalent.“Binding selectively” refers to the situation in which one member of aspecific intra or inter species binding pair will not show anysignificant binding to molecules other than its specific intra or interspecies binding partner (e.g., an affinity of about 100-fold less).Binding is mediated through hydrogen bonding or other molecular forces.“Detectable label” or “Label” means a chemical used to facilitateidentification and/or quantitation of a target substance. Illustrativelabels include labels that can be directly observed or measured orindirectly observed or measured. Such labels include, but are notlimited to, radiolabels that can be measured with radiation-countingdevices; pigments, dyes or other chromogens that can be visuallyobserved or measured with a spectrophotometer; chemiluminescent labelsthat can be measured by a photomultiplier-based instrument orphotographic film, spin labels that can be measured with a spin labelanalyzer; and fluorescent moieties, where the output signal is generatedby the excitation of a suitable molecular adduct and that can bevisualized by excitation with light that is absorbed by the dye or canbe measured with standard fluorometers or imaging systems, for example.The label can be a luminescent substance such as a phosphor orfluorogen; a bioluminescent substance; a chemiluminescent substance,where the output signal is generated by chemical modification of thesignal compound; a metal-containing substance; or an enzyme, where thereoccurs an enzyme-dependent secondary generation of signal, such as theformation of a colored product from a colorless substrate or aspontaneously chemiluminescent product from a suitable precursor. Theterm label can also refer to a “tag” or hapten that can bind selectivelyto a labeled molecule such that the labeled molecule, when addedsubsequently, is used to generate a detectable signal. For instance, onecan use biotin as a tag and then use an avidin or streptavidin conjugateof horseradish peroxidase (HRP) to bind to the tag, and then use achromogenic substrate (e.g., tetramethylbenzidine) or a fluorogenicsubstrate such as Amplex Gold reagent, or a fluorescent tyramide(Molecular Probes, Inc.) to detect the presence of HRP. In a similarfashion, the tag can be a hapten or antigen (e.g., digoxigenin or anoligohistidine), and an enzymatically, fluorescently, or radioactivelylabeled antibody can be used to bind to the tag. Numerous labels areknown by those of skill in the art and include, but are not limited to,microparticles, fluorescent dyes, haptens, enzymes and theirchromogenic, fluorogenic and chemiluminescent substrates and otherlabels that are described in the MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS by Richard P. Haugland, 6^(th)Ed., (1996), and its subsequent 7^(th) edition and 8^(th) editionupdates issued on CD Rom in November 1999 and May 2001, respectively,the contents of which are incorporated by reference, and in otherpublished sources.“Counterstain” (as a verb) means to label all or substantially all ofthe poly(amino acids) that are present in the immobilized poly(aminoacids).“Counterstain” (as a noun) means a reagent or combination of reagentsthat labels all or substantially all of the poly(amino acids) that arepresent in the immobilized poly(amino acids).“Detectable response” means a change in, or occurrence of, a signal thatis detectable either by observation or instrumentally. Typically thedetectable response is an optical response resulting in a change in thewavelength distribution patterns or intensity of absorbance orfluorescence or a change in light scatter, fluorescence lifetime,fluorescence polarization, or a combination of the above parameters.Other detectable responses include, for example, chemiluminescence,phosphorescence, radiation from radioisotopes, attraction to a magnetand electron density.“Dipyrromethenoboron difluoride reactive dyes” means any of chemicallyreactive derivatives of the dyes described in U.S. Pat. Nos. 4,774,339;5,187,288; 5,248,782; 5,274,113; and 5,451,663 that have an absorptionmaximum below 640 nm. Many dipyrrometheneboron difluoride reactive dyesare commercially available under the trademark BODIPY (Molecular Probes,Inc.).“Chemically reactive derivatives” means those dye derivatives thatcovalently label materials under mild conditions.“Chemiluminescent substrate” is defined as any compound which entersinto a chemical reaction with a peroxide or a phosphatase component toproduce chemiluminescence. Typically chemiluminescence is initiated uponan event, such as cleavage of a bond which generates an unstableintermediate that fragments and releases light or a photon as part ofthe high-energy state decay process (U.S. Pat. Nos. 4,931,223 and4,962,192).“Gel electrophoresis” as used herein, refers to a variety of gels thatmay be used in the technique of gel electrophoresis and includes gelsformed by a variety of gel matrix materials, including polyacrylamide,agarose, polyacrylamide-agarose composites, and the like. It alsoencompasses the net migration of a solute under the influence of anelectric field by the combined effects of electroosmosis andelectrophoresis. It further includes any non-denaturing cathodic oranodic electrophoresis, including sodium dodecyl sulfate (SDS)-gelelectrophoresis, isoelectric focusing and gradient gels.“Enzyme” means a protein molecule produced by living organisms, orthrough chemical modification of a natural protein molecule, thatcatalyses chemical reactions of other substances without itself beingdestroyed or altered upon completion of the reactions. Examples of othersubstances, include but are not limited to chemiluminescent,chromogenic, or fluorogenic substances.The term “fluorescent substrate” is used to describe a substrate thatwill produce a fluorescent product upon modification.“Immobilized” means attached to or operatively associated with aninsoluble and/or dehydrated substance or solid phase that comprises oris attached to a solid support.“Kit” means a packaged set of related components, typically one or morecompounds or compositions.“Poly(amino acid)” refers to various natural or synthetic compoundscontaining molecules of amino acids of one or more amino substituentslinked by the carboxyl group of one amino acid and the amino group ofanother, including but not limited to proteins, polypeptides,glycoproteins, etc. Poly(amino acids) comprise one or more a targets towhich an antibody, lectin, aptamer, protein-detection reagent, or othersimilar specific binding pair member can bind. Typically, the samplefrom which poly(amino acids) are selected for analysis comprises tissue,a cell or cells, cell extracts, cell homogenates, purified orreconstituted proteins, recombinant proteins, bodily and otherbiological fluids, viruses or viral particles, prions, subcellularcomponents or synthesized proteins. Possible sources of cellularmaterial for such samples include without limitation plants, animals,fungi, protists, bacteria, archae, or cell lines derived from suchorganisms, including animal cells or animal cell lines that are normalor diseased.“Selective protein-staining techniques” means to generate one or moreseparately detectable signals for a specific target within theimmobilized poly(amino acid) sample.“Solid support” means a substrate material having a rigid or semi-rigidsurface. Typically, at least one surface of the substrate will besubstantially flat, although it may be desirable to physically separatecertain regions with, for example, wells, raised regions, etchedtrenches, or other such topology. Solid support materials also includespheres (including microspheres), rods (such as optical fibers) andfabricated and irregularly shaped items. Solid support materials includeany materials that are used as affinity matrices or supports forchemical and biological molecule syntheses and analyses, such as, butare not limited to: poly(vinylidene difluoride) (PVDF), polystyrene,polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand,pumice, polytetrafluoroethylene, agarose, polysaccharides, dendrimers,buckyballs, polyacrylamide, Kieselguhr-polyacrylamide non-covalentcomposite, polystyrene-polyacrylamide covalent composite,polystyrene-PEG [poly(ethylene glycol)] composite, silicon, rubber, andother materials used as supports for solid phase syntheses, affinityseparations and purifications, hybridization reactions, immunoassays andother such applications. The solid support may be particulate or may bein the form of a continuous surface, such as a microtiter dish or well,a glass slide, a silicon chip, a nitrocellulose sheet, nylon mesh, orother such materials.“Solvent resistant material” means a solid support that is insoluble andinert to solvents commonly utilized in the field of proteomics. Thesolid support would preferably be inert to solvents that include but arenot limited to dimethylformamide, methanol, ethanol, propanol, methylenechloride and N-methyl-2-pyrrolidone.“Target” means a substance of analytical interest that is analyticallydistinguishable from its surrounding environment.“Washing” means contacting a material with a solution to remove ordilute a composition introduced in a previous step. Washing solutionsare typically pure solvents or mixtures of solvents, optionally withsalts or pH-modifying agents that solubilize and/or rinse away some orall of the composition introduced in the previous step.

Selection of Dipyrrometheneboron Difluoride Dyes

In this method, immobilized poly(amino acids) are labeled by incubatingthe immobilized poly(amino acids) with a labeling mixture that comprisesa reactive dipyrrometheneboron difluoride dye. An extensive assortmentof dipyrrometheneboron difluoride dyes and the effects of varioussubstituents on their spectral properties have been described previously(U.S. Pat. Nos. 4,774,339; 5,274,113; 5,187,288; 5,248,782; 5,338,854and 5,433,896, all incorporated by reference). These dyes differ intheir absorption and emission wavelengths; however, the preferred dyeshave substituents that yield dyes with absorption maxima between 495 nmand 640 nm and extinction coefficients >50,000 cm⁻¹M⁻¹.

In one aspect of the invention, the dipyrrometheneboron difluoride dyeshave the formula:

wherein each of R¹ through R⁷ are independently selected from the groupconsisting of H, halogen, L-Rx, and C₁-C₆ alkyl, aryl, arylethenyl,arylbutadienyl, and heteroaryl. By alkyl is meant a saturatedhydrocarbon chain that is optionally further substituted by carboxylicacid, sulfonic acid, or halogen. By aryl is mean an unsaturated 5- or6-membered hydrocarbon ring. By heteroaryl is meant an unsaturated 5- or6-membered ring that contains one or two heteroatoms. Each aryl orheteroaryl ring is optionally further substituted by C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid,or halomethyl. For the preferred dyes, one or more of R¹ through R⁷ isH, two or more of R¹ through R⁷ is nonhydrogen, and only one of R¹through R⁷ is -L-Rx. The element L is a spacer having 1-24 nonhydrogenatoms selected from the group consisting of C, N, O, P, and S and iscomposed of any combination of single, double, triple or aromaticcarbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, andphosphorus-nitrogen bonds. The element Rx is a reactive group that is amaleimide or a succinimidyl ester of a carboxylic acid. In one aspect,-L-Rx is at R¹. In another aspect, -L-Rx is at R². In yet anotheraspect, -L-Rx is at R⁴. In yet another aspect, R¹ is methyl or -L-Rx. Ina further aspect, R² is H, bromine, or -L-Rx. Typically, R³ is H ormethyl. Typically, R⁴ is H or -L-Rx; In preferred embodiments, R⁵ is H,methyl or phenyl. Typically, R⁶ is H or bromine. In preferredembodiments, R⁷ is methyl, phenyl, alkoxyphenyl, phenylethenyl,phenylbutatdienyl pyrrolyl, or thienyl. Preferred embodiments of -L arewhere -L- is —(CH₂)₂—, —(CH₂)₄—, —OCH₂C(O)NH(CH₂)₅—,—(CH₂)₂—C(O)NH(CH₂)₅—, or —(CH)₂C₆H₄OCH₂C(O)NH(CH₂)₅—. Preferably, Rx isa succinimidyl ester of a carboxylic acid.

Dipyrrometheneboron difluoride dyes that are halogenated are notpreferred since halogenation has been shown to reduce the fluorescenceyield of the dye. Dipyrrometheneboron difluoride dyes that aresubstituted at positions 1, 2, 3, 5, 6, and 7 only by hydrogen atoms oralkyl groups tend to have green to yellow-green fluorescence and areoptimally excited by the 488-nm spectral line of the argon-ion laser.Substitution of dipyrrometheneboron difluoride dyes by alkenyl,polyalkenyl, aryl and heteroaryl moieties or combinations of thesesubstituents causes a red-shift of both the absorption and emissionmaxima of the fluorophore, permitting use of the dyes alone or incombination with other labels that have contrasting optical properties.

In a preferred embodiment, the dipyrrometheneboron difluoride reactivedye is an amine-reactive dye since aliphatic amines are common to mostor all proteins. In a more preferred embodiment, the dipyrrometheneborondifluoride is a succinimidyl ester and, in the most preferredembodiments the succinimidyl ester is separated from the fluorophore byan additional seven-atom aminohexanoyl (“X”) spacer. A number ofchemically reactive dipyrrometheneboron dyes are commercially availableunder the trademark BODIPY® dyes, presently including all of thoselisted in Table 1. Properties of these dyes are described by Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (6^(th) Ed.Molecular Probes, Inc., Eugene, Oreg., 1996), which is incorporated byreference. Example 29 provides the procedure used to screen the dyesrepresented in Table 1 for their suitability for detecting total-proteincontent on a blot.

TABLE 1 Reactive dipyrrometheneboron difluoride dyes that are preferredfor this invention. Commercial Dipyrrometheneboron difluoride reactivedyes Product*6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- BODIPYFL-X, SE propionyl)amino)hexanoic acid, succinimidyl ester6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a- BODIPYTMR-X, SE diaza-s-indacene-2-propionyl)amino)hexanoic acid, succinimidylester6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-BODIPY TR-X, SE yl)phenoxy)acetyl)amino) hexanoic acid, succinimidylester 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3- BODIPYR6G-X, SE propionyl)amino)hexanoic acid, succinimidyl ester4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic BODIPYR6G, SE acid, succinimidyl ester6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)BODIPY 630/650-X, styryloxy)acetyl)aminohexanoic acid, succinimidylester SE4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicBODIPY 530/550, acid, succinimidyl ester SE4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicBODIPY 558/568, acid, succinimidyl ester SE4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid,BODIPY 564/570, succinimidyl ester SE4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicBODIPY 576/589, acid, succinimidyl ester SE4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s- BODIPY581/591, indacene-3-propionic acid, succinimidyl ester SE4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8- BODIPY493, 503, propionic acid, succinimidyl ester SE4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicBODIPY FL C5, SE acid, succinimidyl ester6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)BODIPY 650/665-X, styryloxy)acetyl)aminohexanoic acid, succinimidylester SE 2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-BODIPY FL Br₂, SE indacene-3-propionic acid, succinimidyl ester4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicBODIPY FL, SE acid, succinimidyl ester *Available as of the filing datefrom Molecular Probes, Inc., www.probes.com.

The dipyrrometheneboron difluoride dye concentration in the labelingmixture is typically between 1 μM and 100 μM, preferably between about 5μM and about 20 μM; more preferably at least 10 μM.

The synthesis of dipyrrometheneboron difluoride dyes is well documentedin U.S. Pat. Nos. 4,774,339; 5,274,113; 5,187,288; 5,248,782; 5,338,854and 5,433,896, all incorporated by reference, and other publications.Many dyes useful for the invention are available from Molecular Probes,Inc. (Eugene, Oreg.).

The dipyrrometheneboron difluoride dye-labeled immobilized poly(aminoacid) are illuminated to give an optical response that is a fluorescenceemission (fluorescence response). Illumination is by a light sourcecapable of exciting the dipyrrometheneboron difluoride dye-poly(aminoacid) complex, typically at or near the wavelength of an absorptionmaximum, such as an ultraviolet (254-370 nm) or visible (495-640 nm)wavelength emission lamp, an arc lamp, a laser, or even sunlight orordinary room light. Preferably, the sample is excited with a wavelengthwithin 20 nm of the maximum absorption of the dipyrrometheneborondifluoride dye-poly(amino acid) conjugate.

Preferably, the dipyrrometheneboron difluoride dye-poly(amino acid)complexes possess an absorption maximum between 480 and 650 nm, morepreferably between 495 and 640 nm, most preferably matching thewavelength of a laser-based illumination source. The complexes alsopreferably excite with some efficiency in the UV at or near 300 nm.

METHODS OF THE INVENTION

The methods of the invention use chemically reactive dipyrometheneborondifluoride dyes to accomplish counterstaining poly(amino acids) that aresupported of a solid support, wherein the solid supports include but arenot limited to PVDF, nitrocellulose, polystyrene, glass and solidsupports used in microarray and aptamer technologies. In one aspect ofthe invention, the counterstain is used in conjunction with selectiveprotein-staining techniques to generate one or more separatelydetectable signals.

In one embodiment, the method of labeling immobilized poly(amino acids)with dipyrrometheneboron difluoride dyes comprises the steps of:

a. separating poly(amino acids) by gel electrophoresis

b. transferring said separated poly(amino acids) to a solid support,resulting in immobilized poly(amino acids)

c. combining said immobilized poly(amino acids) on said solid supportwith a labeling mixture that comprises one or more dipyrrometheneborondifluoride dyes for a sufficient time for the dyes to form a covalentbond with said poly(amino acids)

The method optionally comprises a step to remove excess and unreacteddipyrrometheneboron difluoride dye. Typically, the excess unreacted dyeand any of its decomposition products are removed by washing.

The poly(amino acids) that are suitable for staining using this methodinclude both synthetic and naturally occurring poly(amino acids),comprising both natural and unnatural amino acids. The poly(amino acids)of the invention include peptides, polypeptides and proteins. Poly(aminoacids) that are labeled and analyzed according to the present methodoptionally incorporate non-peptide regions (covalently ornon-covalently) including lipid (lipopeptides and lipoproteins),phosphate (phosphopeptides and phosphoproteins), and/or carbohydrate(glycopeptides and glycoproteins) regions; or incorporate metal chelatesor other prosthetic groups or non-standard side chains; or aremulti-subunit complexes, or incorporate other organic or biologicalsubstances, such as nucleic acids or cofactors. The poly(amino acids)are optionally relatively homogeneous or heterogeneous mixtures ofpoly(amino acids). Typically, the poly(amino acids) are proteins.

In one embodiment of the invention, the poly(amino acids) areimmobilized on a membrane, such as a poly(vinylidene difluoride) (PVDF)membrane, wherein the poly(amino acids) are applied to the membrane byblotting, spotting, electroblotting or other methods.

In one embodiment of the invention, separated poly(amino acids) inelectrophoretic gels are transferred to a filter membrane or blot orother solid or semi-solid matrix before being combined with the labelingmixture. The present method is effective for both denaturing andnon-denaturing gels. Denaturing gels optionally include a detergent suchas SDS or other alkyl sulfonate (e.g., 0.05%-0.1% SDS). Typically,polyacrylamide or agarose gels are used for electrophoresis. Commonlyused polyacrylamide gels include but are not limited to Tris-glycine,Tris-tricine, mini- or full-sized gels. Agarose gels include modifiedagaroses. Alternatively, the gel is an isoelectric focusing gel orstrip. In addition to polyacrylamide and agarose gels, suitableelectrophoresis gels are optionally prepared using other polymers, suchas HYDROLINK. Alternatively, the electrophoretic gel is a gradient gel.Useful electrophoretic gels for the present invention are eitherprepared according to standard procedures or are purchased commercially.

In another embodiment of the method a specific binding pair member thatbinds selectively to its complementary member is used to detect a targetor targets within the poly(amino acids) that is the complementarymember. Typically, the specific binding pair member contains a label,such that when the binding pair member selectively binds to itscomplementary member, it forms a label-specific binding pair-targetcomplex. In one aspect, the specific binding pair member is covalentlylabeled with an enzyme, and the enzyme is capable utilizing achromogenic, fluorogenic or chemiluminescent substrate to generate adetectable optical response. The specific binding pair member that iscovalently labeled with an enzyme then selectively binds to thetarget/complementary member to form an enzyme-specific bindingpair-target complex.

In general, an enzyme-mediated technique use an enzyme labeled attachedto one member of a binding pair or series of binding pairs as a reagentto detect the complimentary member of the pair or series. In thesimplest case, only the members of one binding pair are used. One memberof the specific binding pair is the analyte, i.e. substance ofanalytical interest. An enzyme is attached to the other (complimentary)member pair, forming a complimentary complex. The complimentaryconjugate attaches to its complimentary analyte to form a complimentarybinding complex. The complimentary binding complexes include but are notlimited to, antibody-antigen interaction, avidin (streptavidin andderivatives thereof) and biotin (including desthiobiotin andiminobiotin), lectins and carbohydrates, immunoglobulins and protein A,G, L or hybrids thereof. Examples 1-5, 8-10, and 15-22 provideexperimental procedures for these methods but these techniques are wellknown to one skilled in the art.

Alternatively, multiple binding pairs may be sequentially linked to thetarget complement in the poly(amino acids), the specific binding pairmember, or to both, resulting in a series of specific binding pairsinterposed between the target and the detectable enzyme label of thespecific binding pair member. Example 23-25 provides a typical procedurefor the use of multiple binding pairs.

In a preferred embodiment of this method, an amine-reactivedipyrrometheneboron difluoride dye selected from Table 2 is used as ageneral protein detection reagent. Optionally an enzyme label on aspecific binding pair member that is capable of utilizing a fluorogenic,chromogenic or chemiluminescent substrate to generate a detectableoptical response is used to detect a specific immobilized protein orproteins. In a preferred embodiment the enzyme substrate is afluorogenic substrate; in the most preferred embodiments the enzymesubstrate yields a detectable fluorescent product at or near the site ofbinding of the specific binding pair member to the specific immobilizedprotein or proteins. In one embodiment, the enzyme is alkalinephosphatase or horseradish peroxidase. In a further embodiment, thealkaline phosphatase or horseradish peroxidase is covalently bound to anantibody to mouse IgG or to rabbit IgG or to a lectin. In a furtherembodiment, the fluorogenic substrate is9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), a polyfluorinated xanthine (U.S. Pat. No. 6,162,931,incorporated by reference), a2-(2′-phosphoryloxyphenyl)-4(3H)-quinazolinone derivative such as ELF 97phosphate or ELF 39 phosphate (U.S. Pat. No. 5,443,986, incorporated byreference) or a tyramide (U.S. Pat. Nos. 5,196,306; 5,583,001 and5,731,158, which are incorporated by reference). The quinazolinonederivatives are commercially available from Molecular Probes, Inc. underthe trademark ELF®. In a further embodiment the peroxidase substrate isan Alexa Fluor, Oregon Green, Marina Blue or biotin-XX tyramide(Molecular Probes, Inc.). In an additional embodiment, thechemiluminescent substrate is the BOLD substrate (Intergen), Fluoroblotsubstrate (Pierce Chemical), ECL® substrates (Amersham Biosciences) orCSPD® substrates (Applied Biosystems).

The fluorogenic quinazolinone substrates are combined with theenzyme-specific binding-pair target-complex under conditions suitablefor the formation of a precipitate. Several variations of fluorogenicquinazolinone substrates are suitable for this invention, including ELF97 and ELF 39 phosphates, blot of which are described in U.S. Pat. No.5,443,986, which is incorporated by reference and are commerciallyavailable, as described in the MOLECULAR PROBES HANDBOOK OF FLUORESCENTPROBES AND RESEARCH CHEMICALS by Richard P. Haugland, 6^(th) Ed.,(1996), and its subsequent 7^(th) edition and 8^(th) edition updatesissued on CD Rom in November 1999 and May 2001, respectively, thecontents of which are incorporated by reference, and in other publishedsources. Examples 4, 5, 23, 24, 25 and 32 contain methods relating tothe use of quinazolinone derivatives as a substrate.

The fluorogenic substrate DDAO phosphate is combined with theenzyme-specific binding-pair target-complex under conditions suitablefor formation of the precipitate. The DDAO phosphate substrate (U.S.Pat. No. 4,810,636) is described in the MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS by Richard P. Haugland, 6^(th)Ed., (1996), and its subsequent 7^(th) edition and 8^(th) editionupdates issued on CD Rom in November 1999 and May 2001, respectively,the contents of which are incorporated by reference, and in otherpublished sources. Examples 1-3, 19, 21-22 and 29-30 describe methodsfor DDAO phosphate use and detection.

The fluorogenic tyramide substrates are combined with the horseradishperoxidase-labeled, specific binding pair-labeled target-complex underconditions suitable for the formation of an immobilized product. The useof labeled tyramides is described in U.S. Pat. Nos. 5,196,306 and5,583,001, the contents of which are incorporated by reference, and inother published sources. Examples 15 describes a method for use anddetection of fluorgenic tyramide substrates.

In another embodiment of the method, the specific binding pair member iscovalently labeled with a fluorescent dye. The specific binding pairmember selectively binds to its complementary member/target, to form afluorescent dye-specific binding-pair target-complex. The fluorescentdye is detectably distinct from the dipyrrometheneboron difluoride dyeused to counterstain the immobilized poly(amino acids) but may be adipyrrometheneboron difluoride dye of a different chemical structure.Preferably, however, the fluorescent dye is a xanthene, includingfluorescein- and rhodamine-based dyes or is a carbocyanine, includingCy3 and Cy5 dyes.

In one aspect of the invention, stained solid supports are used toanalyze the composition of complex sample mixtures and additionally todetermine the relative amount of a particular poly(amino acid) in suchmixtures. Stained solid supports are also used to estimate the purity ofisolated poly(amino acids) and to determine the degree of proteolyticdegradation of the immobilized poly(amino acids). In addition,electrophoretic mobility is optionally used to provide a measure of themolecular weight of uncharacterized poly(amino acids) and to analyzesubunit composition for multi-subunit poly(amino acids), as well as todetermine the stoichiometry for the subunits bound in such proteins. Inthe case of isoelectric focusing electrophoresis (IEF), electrophoreticmobility is used to provide a measure of the net molecular chargepossessed by the poly(amino acid).

The two-dimensional electrophoresis portion of the method of thisinvention can be performed according to known procedures, which may varywidely. In a typical procedure, the sample is first given a linearseparation in an elongate or rod-shaped gel, with an electric potentialimposed along the length of the gel. Migration and separation thus occuralong the gel axis until the proteins in the sample are distributedamong zones positioned along the length of the gel. This is followed byplacement of the elongate gel along one edge of a slab gel, and theimposition of an electric potential in a direction lying within theplane of the slab gel and perpendicular to the edge where the elongategel is placed. The proteins from each zone of the elongate gel migrateinto the slab gel in the direction transverse to the axis of theelongate gel. The result is a two-dimensional array of protein spots inthe slab gel. By using one mode of separation or one set of separationconditions in the first dimension (the elongate gel) and a differentmode or set of separation conditions in the second dimension (the slabgel), highly effective separations can be obtained. For example, thefirst mode may be one based on charge, such as isoelectric focusing, andthe second may be based on molecular weight.

Alternatively, the two dimensions of the separation may be based on thesame parameter but may differ in the compositions of the two gels. Thetwo gels may differ in gel concentration or chemical components. As afurther alternative, the two dimensions of the separation may be basedon the same parameter and performed in gels of the same composition andconcentration, but differ in a separation condition, such as a stepwisedifference in pH, for example. Still further alternatives are the use ofa homogeneous gel in one of the two dimensions and a gradient gel in theother, the use of two different protein solubilizers in the twodimensions, or two different concentrations of the same proteinsolubilizer, and the use of a nonchanging buffer system in one dimensionand a changing (gradient, for example) buffer system in anotherdimension.

Transfer of poly(amino acids) from a gel onto a solid surfaces like amembranes can be carried out using several methods such as a vacuum,capillary action or by means of an electric field (electroblotting).

Poly(amino acids) may be obtained from various sources, includingbiological fermentation media and automated protein synthesizers, aswell as prokaryotic cells, eukaryotic cells, virus particles, tissues,and biological fluids. Suitable biological fluids include, but are notlimited to, urine, cerebrospinal fluid, blood, lymph fluids,interstitial fluid, cell extracts, mucus, saliva, sputum, stool,physiological or cell secretions or other similar fluids. In oneembodiment, the poly(amino acids) comprise the proteome of an animalcell, typically a mammalian cell.

One aspect of the invention comprises separation of the poly(aminoacids) from the electrophoretic matrix. Another aspect of the inventionfurther comprises ionization of the poly(amino acids) and theircharacterization by mass spectroscopy, or transfer and subsequentanalysis of the poly(amino acids) by Edman sequencing.

A further embodiment of the invention is a method of detectingpoly(amino acids) comprising the steps of:

a. combining a poly(amino acids) immobilized on a solid support with alabeling mixture that comprises one or more dipyrrometheneborondifluoride dyes and

b. incubating a labeling mixture for a sufficient time to form acovalent bond between the dipyrrometheneboron difluoride dyes and theimmobilized poly(amino acids) to form a dye poly(amino acid) complex;

c. removing any unbound dipyrrometheneboron difluoride dyes;

d. illuminating the dye-poly(amino acid) complex to yield a detectableoptical response;

e. using the detectable optical response to detect the correspondingdye-poly(amino acid) complex.

In one aspect of this method, the poly(amino acids) are immobilized on amembrane. The immobilized poly(amino acids) are typically on a membraneor in an array. In another aspect, the poly(amino acids) are selectivelybound to a corresponding aptamer, which aptamers are immobilized.

In the subject arrays and aptamers, the poly(amino acids) areimmobilized to the surface of a solid support. By immobilized is meantthat the poly(amino acids) maintain their position relative to the rigidsupport under protein-aptamer complex formation and washing conditions.As such, the poly(amino acids) can be non-covalently or covalentlyassociated with the rigid support surface. Examples of non-covalentassociation include non-specific adsorption, specific binding through aspecific binding pair member covalently attached to the support surface,and entrapment in a matrix material, e.g., a hydrated or driedseparation medium. Examples of covalent binding include covalent bondsformed between the poly(amino acid) and a functional group present onthe surface of the rigid support, e.g., OH, where the functional groupmay be naturally occurring or present as a member of an introducedlinking group, as described in greater detail below.

By solid is meant that the support is rigid and does not readily bend,i.e. the support is not flexible. Examples of solid materials that arenot rigid supports with respect to the present invention includemembranes, flexible plastic films, and the like. As such, the solidsupport of the subject arrays and aptamers are sufficient to providephysical support and structure to the polymeric targets present thereonunder the assay conditions in which the array is employed, particularlyunder high-throughput handling conditions.

The solid support upon which the subject patterns of poly(amino acids)are presented in the subject arrays or aptamers may take a variety ofconfigurations, ranging from simple to complex, depending on theintended use of the array. Thus, the support could have an overall slideor plate configuration, such as a rectangular or disc configuration,where an overall rectangular configuration, as found in standardmicroplates and microscope slides, is preferred. Generally, the lengthof the rigid supports will be at least about 1 cm and may be as great as40 cm or more, but will usually not exceed about 30 cm and may often notexceed about 15 cm. The width of the rigid support will generally be atleast about 1 cm and may be as great as 30 cm, but will usually notexceed 20 cm and will often not exceed 10 cm. The height of the solidsupport will generally range from 0.01 mm to 10 mm, depending at leastin part on the material from which the solid support is fabricated andthe thickness of the material required to provide the requisiterigidity.

The solid support of the subject arrays or aptamers may be fabricatedfrom a variety of materials. The materials from which the substrate isfabricated should ideally exhibit a low level of non-specific binding ofspecific binding pair members during protein-aptamer complex formationor specific binding events. In many situations, it will also bepreferable to employ a material that is transparent to visible and/or UVlight. Specific materials of interest include: glass; plastics, e.g.,polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like; metals, e.g., gold, platinum, and thelike; etc.

The solid support of the subject arrays or aptamers comprise at leastone surface on which the array or aptamers are present, where thesurface may be smooth or substantially planar, or have irregularities,such as depressions or elevations. The surface may be modified with oneor more different layers of compounds that serve to modulate theproperties of the surface in a desirable manner. Such modificationlayers, when present, will generally range in thickness from amonomolecular thickness to about 1 mm, usually from a monomolecularthickness to about 0.1 mm and more usually from a monomolecularthickness to about 0.001 mm. Modification layers of interest include:inorganic and organic layers such as metals, metal oxides, polymers,small organic molecules and the like. Polymeric layers of interestinclude layers of: peptides, proteins, poly(nucleic acids) or mimeticsthereof, e.g., peptide nucleic acids and the like; polysaccharides,phospholipids, polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,polyimides, polyacetates, and the like, where the polymers may behetero- or homopolymeric, and may or may not have separate functionalmoieties attached thereto, e.g., conjugated.

Protein microarray production is a highly automated process, usingeither pin-based or microdispensing liquid handling robots to arrangebiological samples on a flat surface for to generate high-densityprotein arrays and microarrays. This method involves using griddingrobots that transfer either DNA or the corresponding protein expressed,from microplates on to poly(vinylidene difluoride) (Hybond-PVDF,Amersham) membranes in high-density grids. Protein microarrays can alsobe generated by spotting the purified protein from liquid expressioncultures using a transfer stamp mounted onto a flat-bed spotting robot.In addition, there have been many other developments in this area, whichhave been recently reviewed. Such developments include the generation oflow-density protein arrays on filter membranes, such as the universalprotein array system (UPA). This is based on the 96-well microplateformat. In this system, protein microarrays have been printed on anoptically flat glass plate containing 96 wells formed by an enclosinghydrophobic Teflon mask. Inside the wells, arrays of 144 elements each,were spotted using a 36-capillary-based print head attached to aprecise, high-speed, X-Y-Z robot. Standard ELISA techniques and ascanning CCD detector were used for imaging of arrayed antigens. Otherapproaches to protein microarrays that have been reported use eitherphotolithography of silane or gold monolayers, combining microwells withmicrosphere sensors or ink-jetting onto polystyrene film. Such formatsinvolve generating miniaturized immunoassay formats by patterning ofsingle proteins (e.g., BSA, avidin or monoclonal antibodies). Thecurrent invention will aid in the quality control and detection ofproteins immobilized on the various surfaces.

An optical response is detected qualitatively, or optionallyquantitatively, by means that include visual inspection, CCD cameras,video cameras, photographic film, or the use of instrumentation such aslaser scanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. Recording the optical response usingPOLAROID film results in enhanced sensitivity of signal. Responses toother detectable labels (radioactivity, electron spin, magneticproperties etc) is determined with instrumentation appropriate to thelabel.

Applications

The instant invention has useful applications in basic proteomicresearch applications, including but not limited to, 2-D gels incombination with Western blotting, aptamer technology, high-throughputscreening, microarray technology, drug development, and medicaldiagnostics. The methods and kits of the invention can be used in avariety of assay formats for diagnostic applications in the disciplinesof microbiology, immunology, hematology and blood transfusion. Ingeneral, the methods and kits of the current invention provide aversatile and convenient means to enhance the sensitivity andquantitative aspects of any assay that uses immobilized poly(aminoacids) as part of its methodology.

The combination of Western blotting and high-resolution 2-D gelelectrophoresis represents a rapid and simple method toward theidentification of protein spots in complex mixtures, such as lysatesfrom organs, tissues, cells, and body fluids. Frequently the 2-D gelelectrophoresed proteins are transferred onto a solid support(blotting). These “blotted proteins” are analyzed for their antigenicproperties (immunoblotting) using antibodies, though otherprotein-selective probes such as DNA, RNA, labeled aptamers,protein-binding ligands such as fluorescent penicillin analogs forpenicillin-binding proteins and lectins can be used. To obtain areference point in order to identity the protein of interest, a totalprotein pattern from the 2-D gel is required.

The instant invention can be used in conjunction with methods used tostudy poly(amino acids) as a diagnostic or prognostic using aptamers.Aptamers are DNA or RNA molecules that bind specific proteins. Thespecificity of aptamers allows them to distinguish between even closelyrelated proteins, a key advantage for large-scale comprehensivediagnostics and for research proteomics. In addition to the superiorspecificity and affinity of aptamers, their advantages over currentcapture agent technologies include direct detection and cost efficiency.The use of aptamers allows screening of bodily fluids. For example,arrays of aptamers are bound to a solid support, and a sample isapplied. The sample can be any biological sample including but notlimited to blood or urine. Unbound protein is washed off, and theinstant inventions is used to label that label proteins that are boundto the aptamer but not nucleic acids used to generate protein profiles.The instant invention can distinguish functional groups of amino acidsfrom those of nucleic acids and will give a direct readout of proteinson the solid support, even in the presence of nucleic acids, which areessentially unreactive to the preferred amine-reactive dyes of thisinvention. Conversely, labeled aptamers can be used as protein-selectivedetection reagents for immobilized proteins.

The major advantages of using aptamers in high-throughput screeningassays are speed of aptamer identification, high affinity of aptamersfor protein targets, relatively large aptamer-protein interactionsurfaces, and compatibility with various labeling/detection strategies.Aptamers may be particularly useful in high-throughput screening assayswith protein targets that have no known binding partners such as orphanreceptors. Since aptamers that bind to proteins are often specific andpotent antagonists of protein function, the use of aptamers for targetvalidation can be coupled with their subsequent use in high-throughputscreening. Given the large size of many conventional and combinatoriallibraries and the rapid increase in the number of possible therapeutictargets, the speed with which efficient high-throughput screening assayscan be developed can be a rate-limiting step in the discovery process.Applications of the methods and kits of the current invention fordetection of protein binding to immobilized aptamer arrays are expectedto have the same advantages that they do for detection of proteinsimmobilized on blots since the same light sources are used in arrayreaders as in commercially available blot readers.

Proteomics will also play an important role for drug discovery anddevelopment. Proteomics is the link between genes, proteins and disease.Many of the best-selling drugs either act by targeting proteins or areproteins. In addition, many molecular markers of disease, the basis ofdiagnostics, are proteins whose patterns of expression can be used as aguide to drug design. Application of proteomics to study underlyingpharmaceutical mechanisms and to use this information for drugdevelopment is referred to as pharmaceutical proteomics. Unlikeclassical genomic approaches that discover genes related to a disease,proteomics could characterize the disease process directly by findingsets of proteins (pathways or clusters) that participate together incausing the disease. The same technology is used to study the effects ofcandidate drugs intended to reverse a disease process.

EXAMPLES

The following examples describe specific aspects of the invention toillustrate the invention and to provide a description of the methods forthose of skill in the art. The examples should not be construed aslimiting the invention, as the examples merely provide specificmethodology useful in understanding and practicing the invention. Thereagents employed in the examples are commercially available or can beprepared using commercially available instrumentation, methods, orreagents known in the art. The foregoing examples illustrate variousaspects of the invention and practice of the methods of the invention.Each of the references cited in the examples is incorporated herein byreference in its entirety. The examples are not intended to provide anexhaustive description of the many different embodiments of theinvention nor to limit the selection of suitable dyes beyond what hasalready been described above. Thus, although the forgoing invention hasbeen described in some detail by way of illustration and example forpurposes of clarity of understanding, those of ordinary skill in the artwill realize readily that many changes and modifications can be madethereto without departing from the spirit or scope of the appendedclaims.

1. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein Using BODIPY FL-X Succinimidyl Ester and DDAO Phosphate.

A mixture of a dilution series of tubulin (500 ng/lane-0.244 ng/lane)and a constant 250 ng/lane broad range markers (including myosin,β-galactosidase, phosphorylase b, serum albumin, ovalbumin, carbonicanhydrase, trypsin inhibitor, lysozyme and aprotinin) were separated bySDS-polyacrylamide gel electrophoresis utilizing a 4% T, 2.6% C stackinggel, pH 6.8 and a 13% T, 2.6% C separating gel, pH 8.8 according tostandard procedures. % T is the total monomer concentration(acrylamide+crosslinker) expressed in grams per 100 mL and % C is thepercentage crosslinker (e.g., N,N′-methylene-bis-acrylamide,N,N′-diacryloylpiperazine or other suitable agent). Afterelectrophoresis, proteins were transferred to a poly(vinylidenedifluoride) (PVDF) membrane by electroblotting. The membranes wereallowed to dry to minimize loss of proteins during subsequentmanipulations. Membranes were equilibrated by incubation for 10 minutes(two times) in 10 mM sodium borate buffer, pH 9.5. After equilibration,the membranes were stained for 30 minutes with 10 □M BODIPY FL-X,succinimidyl ester in the same buffer. After two 5-10 minute washes in100% methanol, the membranes were rinsed with dH₂O and allowed to airdry. Immunodetection was performed by standard procedures, including a 1hour blocking step, 2 hour primary antibody incubation, and 1 houralkaline phosphatase-conjugated secondary antibody incubation at roomtemperature with agitation. The blocking buffer contained 0.25% MOWIOL4-88, 0.5% BSA and 0.2% Tween 20. To detect the presence of the alkalinephosphatase-conjugated secondary antibody on the blot, the membraneswere incubated for 30 minutes with 1.25 μg/mL DDAO phosphate in a buffercontaining 1 mM MgCl₂ and 10 mM Tris, pH 9.5. Proteins were viewed usinga 300 nm UV epi-illuminator. The total protein profile appeared greenfluorescent while the specific target was stained red fluorescent.Membranes can also be imaged using a laser system such as the FujiFLA-3000 imager utilizing the 633 nm excitation filter and 675 nmemission filter for the DDAO dye and a 473 nm excitation filter and 520nm emission filter for the BODIPY FL-X dye. Other BODIPY dyes can beutilized similarly.

2. Serial Dichromatic Detection of a Specific Target Using DDAOPhosphate Followed by Total Protein Detection Using BODIPY FL-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted by standard methods. The membranes were allowed to dry tominimize loss of proteins during subsequent manipulations.Immunodetection was performed by standard procedures. To detect thepresence of the alkaline phosphatase-conjugated secondary antibody onthe blot the membranes were incubated for 30 minutes with 1.25 μg/mLDDAO phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH 9.5.Viewing membranes by UV illumination or using a 633 nm helium-neon laserscanner revealed a red-fluorescent signal associated with the targetprotein. The membranes were allowed to dry and were then equilibrated byincubation for 10 minutes (two times) in 10 mM sodium borate buffer, pH9.5. After equilibration, the membranes were stained for 30 minutes with10 □M BODIPY FL-X, succinimidyl ester in the same buffer. After 5-10minute washes in 100% methanol the membranes were rinsed with dH₂O andallowed to dry. Proteins were viewed using a 300 nm UV epi-illuminator.The total protein profile appeared green fluorescent while the specifictarget was not detected because the DDAO dye does not precipitate on themembrane and was washed off during subsequent staining steps. OtherBODIPY dyes can be utilized similarly.

3. Stripping Dichromatic Membranes of the DDAO Signal to AllowRe-Probing with Another Antibody.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andsubsequently electroblotted by standard methods, as described in theprevious examples. The membranes were allowed to dry to minimize loss ofproteins during subsequent manipulations. Membranes were equilibrated byincubation for 10 minutes (two times) in 10 mM sodium borate buffer, pH9.5. After equilibration, the membranes were stained for 30 minutes with10 □M BODIPY FL-X, SE in the same buffer. After 5-10 minute washes in100% methanol, the membranes were rinsed with dH₂O and allowed to dry.Immunodetection was performed by standard methods, as described in theprevious examples. To detect the presence of the alkalinephosphatase-conjugated secondary antibody on the blot the membranes wereincubated for 30 minutes with 1.25 μg/mL DDAO phosphate (MolecularProbes, Inc, Eugene, Oreg.) in a buffer containing 1 mM MgCl₂ and 10 mMTris, pH 9.5. Proteins were viewed using a 300 nm UV epi-illuminator.The total protein profile appeared green fluorescent while the specifictarget was stained red fluorescent. After initial verification ofprotein labeling, the membranes were stripped by incubating them for 1hour, at 500, in buffer containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8 and50 mM DTT. After rinsing the membranes with 50 mM Tris, pH 7.5 and 150mM NaCl, proteins previously detected with the DDAO dye were no longervisible using a 300 nm UV epi-illuminator. The total protein profilestill appeared green fluorescent. These membranes could be incubatedwith another monoclonal antibody and an alkaline phosphatase- orhorseradish peroxidase-conjugated secondary antibody in combination withsuitable enzyme substrates, permitting detection of a different antigenon the blot. The green-fluorescent BODIPY FL dye signal remained, evenafter the stripping and re-probing steps. Other BODIPY dyes can beutilized similarly.

4. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein Using BODIPY TR-X Succinimidyl Ester and ELF 97Phosphate.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted by standard methods, as described in Example 1. Themembranes were allowed to dry to minimize loss of proteins duringsubsequent manipulations. Membranes were equilibrated by incubation for10 minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 □MBODIPY TR-X, SE in the same buffer. After two 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry.Immunodetection was performed by standard procedures. To detect thepresence of the alkaline phosphatase-conjugated secondary antibody onthe blot the membranes were incubated for 30 minutes with 10 μg/mL ELF97 phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH 9.5.Proteins were viewed using 300 nm UV epi-illumination. The total proteinprofile appeared red fluorescent while the specific target was stainedgreen fluorescent. Labeling with the ELF 97 phosphate substrate prior tolabeling with BODIPY TR-X succinimidyl ester produced the same results.Other BODIPY dyes can be utilized similarly.

5. Dichromatic Detection Using BODIPY TR-X Succinimidyl Ester and ELF 97Phosphate Followed by Stripping of the Membranes for Re-Probing.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were allowed todry to minimize loss of proteins during subsequent manipulations.Membranes were equilibrated by incubation for 10 minutes (two times) in10 mM sodium borate buffer, pH 9.5. After equilibration, the membraneswere stained for 30 minutes with 10 □M BODIPY TR-X, SE in the samebuffer. After 5-10 minute washes in 100% methanol the membranes wererinsed with dH₂O and allowed to dry. Immunodetection was performed bystandard procedures. To detect the presence of the alkalinephosphatase-conjugated secondary antibody on the blot the membranes wereincubated for 30 minutes with 10 μg/mL ELF 97 phosphate in a buffercontaining 1 mM MgCl₂ and 10 mM Tris, pH 9.5. Proteins were viewed using300 nm UV epi-illumination. The total protein profile appeared redfluorescent while the specific target was stained green fluorescent.After initial verification of protein labeling, the membranes wereincubated for 1 hour, at 50° C., in buffer containing 2% SDS, 62.5 mMTris-HCl, pH 6.8 and 50 mM DTT. After rinsing the membranes with 50 mMTris, pH 7.5, 150 mM NaCl, proteins previously detected with ELF 97phosphate were still visible using a 300 nm UV epi-illuminator. Thisdemonstrates that the ELF 97 alcohol precipitate was permanent. Thetotal protein profile appeared as red-fluorescent bands. Other BODIPYdyes can be utilized similarly.

6. Simultaneous Dichromatic Detection of a Specific Target UsingBCIP/NBT Followed by Total Protein Detection Using BODIPY TR-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were allowed todry to minimize loss of proteins during subsequent manipulations.Immunodetection was performed by standard procedures. To detect thepresence of the alkaline phosphatase-conjugated secondary antibody onthe blot the membranes were incubated for 3-60 minutes in NBT/BCIP in abuffer containing 100 mM Tris, pH 9.5, 50 mM MgCl₂ and 100 mM NaCl. Theendpoint of incubation was determined by eye, based upon colordevelopment. The membranes were allowed to dry and were thenequilibrated by incubation for 10 minutes (two times) in 10 mM sodiumborate buffer, pH 9.5. After equilibration, the membranes were stainedfor 30 minutes with 10 □M BODIPY TR-X, succinimidyl ester in the samebuffer. After two 5-10 minute washes in 100% methanol the membranes wererinsed with dH₂O and allowed to dry. The total protein profile wasviewed using 300 nm UV epi-illumination and appeared red fluorescentwhile the NBT/BCIP signal associated with the specific target appearedblue colored when using white light to visualize the signal. NBT/BCIPwas normally purple in color but when followed by BODIPY TR-X,succinimidyl ester it appeared blue. Other BODIPY dyes can be utilizedsimilarly.

7. Serial Dichromatic Detection of Total Protein Using BODIPY TR-XSuccinimidyl Ester Followed by a Specific Target Using NBT/BCIP.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were allowed todry to minimize loss of proteins during subsequent manipulations.Membranes were then equilibrated by incubation for 10 minutes (twotimes) in 10 mM sodium borate buffer, pH 9.5. After equilibration, themembranes were stained for 30 minutes with 10 □M BODIPY TR-X,succinimidyl ester in the same buffer. After two 5-10 minute washes in100% methanol the membranes were rinsed with dH₂O and allowed to dry.Proteins appeared as red-fluorescent bands when visualized by UVepi-illumination. Immunodetection was performed by standard procedures.To detect the presence of the alkaline phosphatase-conjugated secondaryantibody on the blot the membranes were incubated for 3-60 minutes inNBT/BCIP in a buffer containing 100 mM Tris, pH 9.5, 50 mM MgCl₂, and100 mM NaCl. The endpoint of incubation was determined by eye based oncolor development. The red fluorescence of the BODIPY dye was quenchedby the NBT/BCIP. The NBT/BCIP precipitate appears purple by eye. OtherBODIPY dyes can be utilized similarly.

8. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Using BODIPY TR-X Succinimidyl Ester and Fluoroblot™ PeroxidaseSubstrate.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY TR-X, SE in the same buffer. After 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry.Immunoblotting was performed by standard procedures including a 1 hourblocking step, 2 hour primary antibody incubation, and 1 hourhorseradish peroxidase-conjugated secondary antibody incubation at roomtemperature with agitation. The blocker included 0.25% MOWIOL 4-88, 0.5%BSA, and 0.2% Tween 20. To detect the presence of the horseradishperoxidase-conjugated secondary antibody on the blot the membranes wereincubated for 30 minutes in Fluoroblot peroxidase substrate (PierceChemical Company, Milwaukee, Wis.) diluted 1:1 in stable peroxidasebuffer. Proteins were viewed using 300 nm UV epi-illumination. The totalprotein profile appeared red fluorescent while the specific target wasstained green fluorescent. The Fluoroblot dye-labeled proteins wereviewed best when the membranes were wet because the fluorescence wasconsiderably reduced upon drying. Other BODIPY dyes can be utilizedsimilarly.

9. Serial Dichromatic Detection of a Specific Target Followed by TotalProtein Detection Using Fluoroblot Peroxidase Substrate and BODIPY Dyes.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Immunodetection was performed by standard procedures. Todetect the presence of the horseradish peroxidase-conjugated secondaryantibody on the blot the membranes were incubated for 30 minutes inFluoroblot peroxidase substrate diluted 1:1 in stable peroxidase buffer.Upon viewing with a 300 nm epi-illumination source, the target proteinappeared green fluorescent. The membranes were allowed to dry and werethen equilibrated by incubation for 10 minutes (two times) in 10 mMsodium borate buffer, pH 9.5. After equilibration, the membranes werestained for 30 minutes with 10 □M BODIPY TR-X, succinimidyl ester in thesame buffer. After 5-10 minute washes in 100% methanol the membraneswere rinsed with dH₂O and allowed to dry. Proteins were viewed using 300nm UV epi-illumination. The total protein profile appeared fluorescentred while the specific target was no longer detected because theFluoroblot dye was washed off the membrane in the subsequent BODIPY dyestaining steps. Other BODIPY dyes can be utilized similarly.

10. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Using BODIPY TR-X Succinimidyl Ester and Amplex Gold Substrate.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 □MBODIPY TR-X, SE in the same buffer. After 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry.Immunoblotting was performed by standard procedures including a 1 hourblocking step, 2 hour primary antibody incubation, and 1 hourhorseradish peroxidase-conjugated secondary antibody incubation at roomtemperature with agitation. The blocker included 0.25% MOWIOL 4-88, 0.5%BSA, and 0.2% Tween 20. To detect the presence of the horseradishperoxidase-conjugated secondary antibody on the blot the membranes wereincubated for 30 minutes with 50□□ Amplex Gold in a buffer containing 1mM MgCl₂, 280 μM ZnCl₂, 10 mM Tris, pH 9.5 and 200 μM H₂O₂. Proteinswere viewed using a 300 nm UV epi-illuminator. The total protein profileappeared red fluorescent while the specific target was labeledgoldenrod. Proteins were viewed using a 300 nm UV epi-illuminator.Membranes could also have been imaged using a laser system such as theFuji FLA-3000 imager utilizing the 633 nm excitation filter and 675 nmemission filter for the BODIPY TR-X dye and a 532 nm excitation filterand 580 nm emission filter for Amplex Gold. Other BODIPY dyes can beutilized similarly.

11. Serial Dichromatic Detection of Total Protein and a Specific TargetUsing BODIPY TR-X Succinimidyl Ester and 4-chloro-1-naphthol.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were allowed todry to minimize loss of proteins during subsequent manipulations.Membranes were then equilibrated by incubation for 10 minutes (twotimes) in 10 mM sodium borate buffer, pH 9.5. After equilibration, themembranes were stained for 30 minutes with 10 □M BODIPY TR-X, SE in thesame buffer. After two 5-10 minute washes in 100% methanol the membraneswere rinsed with dH₂O and allowed to dry. Proteins appeared asred-fluorescent bands upon 300 nm UV epi-illumination. Immunodetectionwas performed by standard procedures. To detect the presence of thehorseradish peroxidase-conjugated secondary antibody on the blot themembranes were incubated for 3-60 minutes in 4-chloro-1-naphtholsolution (0.48 mM 4-chloro-1-naphthol, 50 mM Tris HCl, 0.2 mM NaCl and17% methanol) to which was added 0.01% (v/v) hydrogen peroxide. Theendpoint of incubation was determined by eye based upon colordevelopment. The fluorescence of the total protein profile was quenchedby the 4-chloro-1-naphthol. The 4-chloro-1-naphthol labeling of thespecific target protein appears purple by eye when viewed usingwhite-light illumination. Other BODIPY dyes can be utilized similarly.

12. Simultaneous Dichromatic Detection of a Specific Target Using4-chloro-1-naphthol Followed by Total Protein Staining Using BODIPY TR-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were allowed todry to minimize loss of proteins during subsequent manipulations.Immunodetection was performed by standard procedures. To detect thepresence of the horseradish peroxidase-conjugated secondary antibody onthe blot the membranes were incubated for 3-60 minutes in4-chloro-1-naphthol solution (0.48 mM 4-chloro-1-naphthol, 50 mM TrisHCl, 0.2 mM NaCl and 17% methanol) to which 0.01% (v/v) hydrogenperoxide was added. The membranes were allowed to dry and were thenequilibrated by incubation for 10 minutes (two times) in 10 mM sodiumborate buffer, pH 9.5. After equilibration, the membranes were stainedfor 30 minutes with 10 □M BODIPY TR-X, SE in the same buffer. After two5-10 minute washes in 100% methanol the membranes were rinsed with dH₂Oand allowed to dry. Proteins were viewed using 300 nm UVepi-illumination. The total protein profile appeared red fluorescent.The specific target appears purple when viewed by white-lightillumination. Other BODIPY dyes can be utilized similarly.

13. Serial Dichromatic Detection of Total Protein and a Specific TargetUsing BODIPY TR-X Succinimidyl Ester and 3,3′-diaminobenzidinetetrahydrochloride.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY TR-X, succinimidyl ester in the same buffer. After 5-10 minutewashes in 100% methanol the membranes were rinsed with dH₂O and allowedto dry. Upon viewing with a 300 nm UV epi-illumination source, proteinsappeared as red-fluorescent bands. Immunodetection was performed bystandard procedures. To detect the presence of the horseradishperoxidase-conjugated secondary antibody on the blot the membranes wereincubated for 3-60 minutes with 0.6 mg/mL 3,3′-diaminobenzidine in 50 mMTris, pH 7.6, 0.03% hydrogen peroxide (v/v). The endpoint of incubationwas determined by eye based upon color development. The fluorescence ofthe total protein profile was quenched by the 3,3′-diaminobenzidine. The3,3′-diaminobenzidine labeling the specific target protein appearedbrown on the blot when viewed by white-light illumination. Other BODIPYdyes can be utilized similarly.

14. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Using BODIPY TR-X Succinimidyl Ester and 3,3′-diaminobenzidine.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Immunoblotting was performed by standard procedures. Todetect the presence of the horseradish peroxidase-conjugated secondaryantibody on the blot the membranes were incubated for 3-60 minutes with0.6 mg/mL 3,3′-diaminobenzidine in 50 mM Tris, pH 7.6, 0.03% hydrogenperoxide (v/v). The membranes were allowed to dry and were thenequilibrated by incubation for 10 minutes (two times) in 10 mM sodiumborate buffer, pH 9.5. After equilibration, the membranes were stainedfor 30 minutes with 10 μM BODIPY TR-X, SE in the same buffer. After 5-10minute washes in 100% methanol, the membranes were rinsed with dH₂O andallowed to dry. Proteins were viewed as red-fluorescent bands using 300nm UV epi-illumination. The specific target was labeled brown and waseasily viewed using white-light illumination. Other BODIPY dyes can beutilized similarly.

15. Simultaneous Dichromatic Detection of a Specific Target Using AlexaFluor 488 Tyramide Conjugate and Total Protein Using BODIPY TR-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. Immunodetection was performed by standard procedures. Todetect the presence of the horseradish peroxidase-conjugated secondaryantibody on the blot the membranes were incubated for 30 minutes in 5 μMAlexa Fluor 488 tyramide (Molecular Probes, Inc, Eugene Oreg., U.S. Pat.No. 6,130,101 and U.S. patent Ser. No. 09/969,853, incorporated byreference) in 50 mM Tris, pH 7.5 and 150 mM NaCl to which was added0.03% hydrogen peroxide (v/v). The membranes were allowed to dry andwere then equilibrated by incubation for 10 minutes (two times) in 10 mMsodium borate buffer, pH 9.5. After equilibration, the membranes werestained for 30 minutes with 10 □M BODIPY TR-X, SE in the same buffer.After 5-10 minute washes in 100% methanol the membranes were rinsed withdH₂O and allowed to dry. Proteins can be viewed using 300 nm UVepi-illumination. The total protein profile appeared red fluorescentwhile the specific target appeared green fluorescent. Other BODIPY dyescan be utilized similarly in combination with appropriately selectedfluorescent tyramide derivatives.

16. Simultaneous Dichromatic Detection of Glycoproteins and TotalProteins Using BODIPY TR-X Succinimidyl Ester and PRO-Q EMERALD 300Glycoprotein Stain on Membranes.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. The membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY TR-X, SE in the same buffer. After 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry. Themembranes were then fixed in 50% methanol, oxidized in 1% periodic acidin 3% acetic acid and stained with 5 □M PRO-Q EMERALD 300 in 2% DMF, 250mM MgCl₂, and 3% acetic acid. After staining, the membranes were rinsedin 3% acetic acid followed by dH₂O and allowed to dry. Proteins wereviewed using 300 nm UV epi-illumination. The total protein profileappeared red fluorescent while the glycoproteins were green fluorescent.Labeling with the PRO-Q EMERALD 300 dye prior to labeling with BODIPYTR-X succinimidyl ester produced the same results. Other BODIPY dyes canbe utilized similarly.

17. Simultaneous Dichromatic Detection of Glycoproteins and TotalProteins Using BODIPY TR-X Succinimidyl Ester and PRO-Q EMERALD 300Glycoprotein Stain in Gels.

Proteins were separated by SDS-polyacrylamide gel electrophoresis, asdescribed in Example 1. After electrophoresis, gels were fixed overnightin MeOH:acetic acid:H₂O (5:1:1). They were then washed and equilibratedin 10 mM sodium borate buffer, pH 9.5, and stained in 10 μM BODIPY TR-X,SE in the same buffer. After washing, the gels were fixed in 50%methanol, oxidized in 1% periodic acid in 3% acetic acid and stainedwith 5 □M PRO-Q EMERALD 300 in 2% DMF, 250 mM MgCl₂, and 3% acetic acid.After staining, the gels were rinsed in 3% acetic acid followed by dH₂O.Proteins stained with PRO-Q EMERALD 300 can be viewed using an imagingsystem such as the Roche Lumilmager, which utilizes a UV light box and aCCD camera. PRO-Q EMERALD 300 dye-stained proteins can be visualizedusing the 520 nm+/−20 emission filter. BODIPY TR-X dye-conjugatedproteins were visualized and separated from PRO-Q EMERALD 300dye-stained proteins using the 590/10 excitation filter and a 625/30emission filter.

18. Simultaneous Dichromatic Detection of Glycoproteins Using LectinConcanavalin A, Alkaline Phosphatase Conjugate and BODIPY TR-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. The membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY TR-X, SE in the same buffer. After two 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry. Fordetection of the glycoproteins described above, the membranes wereblocked then incubated in 1 □g/mL concanavalin A-alkaline phosphatase ina buffer containing 150 mM mTBS, 50 Tris, pH 7.5, 0.2% Tween-207, 0.25%Mowiol 4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detection of theconcanavalin A-AP conjugate, the membranes were incubated with 10 □g/mLELF 97 phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH9.5. Proteins were viewed using 300 nm UV epi-illumination. ConcanavalinA binds to glycoproteins containing □-mannosyl and □-glucopyranosylresidues. The total protein profile appeared red fluorescent while thetargeted glycoproteins were stained green fluorescent.

19. Simultaneous Dichromatic Detection of Glycoproteins Using LectinConcanavalin A, Alkaline Phosphatase Conjugate and BODIPY FL-XSuccinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. The membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes in 10 □M BODIPYFL-X, SE in the same buffer. After two 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry. Fordetection of the glycoproteins described above, the membranes wereblocked then incubated in 1 □g/mL concanavalin A-alkaline phosphatase ina buffer containing 150 mM mTBS, 50 Tris, pH 7.5, 0.2% Tween-20, 0.25%Mowiol 4-88, 0.5 mM MgCl₂ and 1 mM CaCl₂. For detection of theconcanavalin A-AP conjugate, the membranes were incubated in 1.25 μg/mLDDAO phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH 9.5.Proteins were viewed using a 300 nm UV epi-illuminator. The totalprotein profile appeared green fluorescent while the targetedglycoproteins were stained red fluorescent. Membranes can also be imagedusing a laser system such as the Fuji FLA-3000 imager utilizing the 633nm excitation filter and 675 nm emission filter for the DDAO dye and a473 nm excitation filter and 520 nm emission filter for the BODIPY FL-Xdye.

20. Simultaneous Dichromatic Detection of Glycoproteins Using LectinWheat Germ Agglutinin and BODIPY TR-X Succinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. The membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY TR-X, SE in the same buffer. After two 5-10 mL washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry. Fordetection of the glycoproteins described above, the membranes wereblocked then incubated with 0.5 μg/mL wheat germ agglutinin-alkalinephosphatase in a buffer containing 150 mM mTBS, 50 Tris, pH 7.5, 0.2%Tween-20, 0.25% Mowiol 4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detectionof the wheat germ agglutinin-AP conjugate, the membranes were incubatedwith 10 μg/mL ELF 97 phosphate in a buffer containing 1 mM MgCl₂ and 10mM Tris, pH 9.5. Proteins were viewed using 300 nm UV epi-illumination.Wheat germ agglutinin binds glycoproteins containing N-acetylglucosamineand N-acetylneuraminic acid residues. The total protein profile appearedred fluorescent while the targeted glycoproteins were stained greenfluorescent.

21. Simultaneous Dichromatic Detection of Glycoproteins Using LectinWheat Germ Agglutinin and BODIPY FL-X Succinimidyl Ester.

Proteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes were thenallowed to dry to minimize loss of proteins during subsequentmanipulations. The membranes were then equilibrated by incubation for 10minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 □MBODIPY FL-X, SE in the same buffer. After two 5-10 minute washes in 100%methanol the membranes were rinsed with dH₂O and allowed to dry. Fordetection of the glycoproteins described above, the membranes wereblocked then incubated with 0.5 μg/mL wheat germ agglutinin-alkalinephosphatase in a buffer containing 150 mM mTBS, 50 Tris, pH 7.5, 0.2%Tween-20, 0.25% Mowiol 4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detectionof the wheat germ agglutinin-AP conjugate, the membranes were incubatedwith 1.25 μg/ml DDAO phosphate in a buffer containing 1 mM MgCl₂ and 10mM Tris, pH 9.5. Proteins can be viewed using a 300 nm UVepi-illuminator. The total protein profile appeared green fluorescentwhile the glycoproteins were stained red fluorescent. Membranes can alsobe imaged using a laser system such as the Fuji FLA-3000 imagerutilizing the 633 nm excitation filter and 675 nm emission filter forthe DDAO dye and a 473 nm excitation filter and 520 nm emission filterfor the BODIPY FL-X dye.

22. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein Using BODIPY FL-X Succinimidyl Ester in 2-DElectrophoresis and DDAO Phosphate.

150 □g of a rat fibroblast lysate was applied to 1 mm diameter, 20 cmlong isoelectric focusing gels consisting of a 4% T, 2.6% Cpolyacrylamide gel matrix, containing 9 M urea, 2% Triton X-100, and 2%carrier ampholytes. Gels were run vertically for 18,000 volt-hours using10 mM phosphoric acid and 100 mM sodium hydroxide as the anode andcathode buffer, respectively. Isoelectric focusing gels were incubatedin 0.3 M Tris base, 0.075 M Tris-HCl, 3% SDS, 0.01% bromophenol blue fortwo minutes. Isoelectric focusing gels were then laid on top of 1 mmthick, 20 cm×20 cm, 12.5% T, 2.6% C polyacrylamide gels containing 375mM Tris-base, pH 8.8 and SDS-polyacrylamide gel electrophoresis wasperformed according to standard procedures except that the cathodeelectrode buffer was 50 mM Tris, 384 mM glycine, 4% sodium dodecylsulfate, pH 8.8 while the anode electrode buffer is 25 mM Tris, 192 mMglycine, 2% sodium dodecyl sulfate, pH 8.8. After the second dimensionelectrophoresis, proteins were transferred to a 20 cm×20 cm piece ofpoly(vinylidene difluoride) (PVDF) membrane by electroblotting. Themembranes were allowed to dry to minimize loss of proteins duringsubsequent manipulations. Membranes were equilibrated by incubation for10 minutes (two times) in 10 mM sodium borate buffer, pH 9.5. Afterequilibration, the membranes were stained for 30 minutes with 10 μMBODIPY FL-X, succinimidyl ester in the same buffer. After two 5-10minute washes in 100% methanol, the membranes were rinsed with dH₂O andallowed to air dry. Immunodetection was performed by standardprocedures, including a 1 hour blocking step, 2 hour primary antibodyincubation, and 1 hour alkaline phosphatase-conjugated secondaryantibody incubation at room temperature with agitation. The blockingbuffer contained 0.25% Mowiol 4-88, 0.5% BSA and 0.2% Tween 20. Todetect the presence of the alkaline phosphatase-conjugated secondaryantibody on the membrane, the membranes were incubated for 30 minuteswith 1.25 μg/mL DDAO phosphate in a buffer that contained 1 mM MgCl₂ and10 mM Tris, pH 9.5. Proteins were viewed using a 300 nm UVepi-illuminator. The total protein profile appeared green fluorescentwhile the specific target was stained red fluorescent. Membranes canalso be imaged using a laser system such as the Fuji FLA-3000 imagerutilizing the 633 nm excitation filter and 675 nm emission filter forthe DDAO dye and a 473 nm excitation filter and 520 nm emission filterfor the BODIPY FL-X dye. Other BODIPY dyes can be utilized similarly.

23. Trichromatic Detection of Two Specific Targets Using ELF 39Phosphate, Alexa Fluor 350 and BODIPY TR-X Succinimidyl Ester in 2-DElectrophoresis.

300 □g of bovine heart mitochondria were diluted to 5 mg/mL into 25 mMTris, 2 mM EDTA, pH 7.5. Just prior to extraction, 1 mM PMSF was added,followed by 1% n-dodecyl-β-D-maltoside for the actual extraction. Themitochondria were incubated in the detergent for 20 minutes before theinsoluble material was pelleted by centrifugation (10 min, 12,000 rpm).The protein in the supernatant was precipitated with 10% TCA andpelleted by centrifugation. The pellet was resuspended in urea samplebuffer (7 M urea, 2 M thiourea, 1% Zwittergent 3-10, 2% CHAPS, 0.8%carrier Ampholytes) and frozen at −20° C. IPG strips (3-10 NL) wererehydrated overnight at room temperature in the same urea buffer (450mL). The IPG strip was placed into the pHaser isoelectric focusingsystem and the sample was applied on a piece of filter paper at theanodic end. The sample was subjected to isolelectric focusing at 20° C.for 24.5 h (70,000 Vh) and then separated on a 20 cm×20 cm×1 mm seconddimension SDS-polyacrylamide gel (12.5% T, 2.6% C).

After second dimension electrophoresis, proteins were transferred to a20 cm×20 cm piece of PVDF membrane by electroblotting. The membraneswere allowed to dry to minimize loss of proteins during subsequentmanipulations. Membranes are equilibrated by incubation for 10 minutes(two times) in 10 mM sodium borate buffer, pH 9.5. After equilibration,the membranes were stained for 30 minutes with 10 □M BODIPY TR-X,succinimidyl ester in the same buffer. After two 5-10 minute washes in100% methanol, the membranes were rinsed with dH₂O and allowed to airdry. Immunodetection was performed by standard procedures except thattwo primary antibodies to two different targets were applied to themembrane. One primary antibody was directly conjugated to the AlexaFluor 350 dye and the other was a mouse monoclonal antibody that wassubsequently detected with a goat anti-mouse-alkaline phosphatasesecondary antibody. To detect the presence of the alkalinephosphatase-conjugated secondary antibody on the blot, the membraneswere incubated for 30 minutes in 10 μg/mL ELF 97 phosphate in a buffercontaining 1 mM MgCl₂ and 10 mM Tris, pH 9.5. Proteins were viewed using300 nm UV epi-illumination. The total protein profile appeared redfluorescent while one specific target was stained green fluorescent andthe other was labeled blue fluorescent.

24. Trichromatic Detection of Two Specific Targets Using ELF 97Phosphate, Streptavidin Alexa Fluor 350 dye, and BODIPY TR-XSuccinimidyl Ester.

A mixture of a dilution series of tubulin (500 ng/lane-0.244 ng/lane),biotin-labeled concanavalin-A (500 ng/lane-0.244 ng/lane) and a constant250 ng/lane broad range markers (including myosin, β-galactosidase,phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsininhibitor, lysozyme and aprotinin) were separated by SDS-polyacrylamidegel electrophoresis and electroblotted as described in Example 1. Themembranes were then allowed to dry to minimize loss of proteins duringsubsequent manipulations. The membranes were then equilibrated byincubation for 10 minutes (two times) in 10 mM sodium borate buffer, pH9.5. After equilibration, the membranes were stained for 30 minutes with10 μM BODIPY TR-X, SE in the same buffer. After two 5-10 mL washes in100% methanol, the membranes were rinsed with dH₂O and allowed to airdry. Immunodetection was performed by standard procedures except thatmembranes were incubated in a mixture of a secondary goat anti-mouseIgG-alkaline phosphatase antibody and streptavidin Alexa Fluor 350. Todetect the presence of the alkaline phosphatase-conjugated secondaryantibody on the membranes, the membranes were incubated for 30 minuteswith 10 μg/mL ELF 97 phosphate in a buffer containing 1 mM MgCl₂ and 10mM Tris, pH 9.5. Proteins were viewed using 300 nm UV epi-illumination.The total protein profile appeared red fluorescent while one specifictarget was stained green fluorescent and the biotinylated proteins werestained blue fluorescent.

25. Trichromatic Detection of Two Specific Targets Using a GoatAnti-Mouse Secondary Antibody Conjugated to Alexa Fluor 350, ELF 97Phosphate, and BODIPY TR-X Succinimidyl Ester.

A mixture of proteins (as in Example 24) was separated bySDS-polyacrylamide gel electrophoresis and electroblotted, as describedin Example 1. The membranes were then allowed to dry to minimize loss ofproteins during subsequent manipulations. The membranes were thenequilibrated by incubation for 10 minutes (two times) in 10 mM sodiumborate buffer, pH 9.5. After equilibration, the membranes were stainedfor 30 minutes with 10 μM BODIPY TR-X, SE in the same buffer. After two5-10 minute washes in 100% methanol, the membranes were rinsed with dH₂Oand allowed to air dry. Immunodetection was performed by standardprocedures, except that membranes were incubated in a mixture of asecondary goat anti-mouse IgG-Alexa Fluor 350 conjugate andstreptavidin-alkaline phosphatase. To detect the presence of thealkaline phosphatase-conjugated streptavidin on the blot the membraneswere incubated for 30 minutes with 10 μg/mL ELF 97 phosphate (U.S. Pat.No. 5,316,906, incorporated by reference) in a buffer containing 1 mMMgCl₂ and 10 mM Tris, pH 9.5. Proteins were viewed using 300 nm UVepi-illumination. The total protein profile appeared red fluorescentwhile one specific target was stained blue fluorescent and thebiotinylated proteins were stained green fluorescent.

26. Simultaneous Two Color Detection with in-Gel Zymography andTotal-Protein Staining Using BODIPY TR-X Succinimidyl Ester.

A partially purified protein sample containing an enzyme of interest,such as β-glucuronidase, was diluted and loaded onto a standardSDS-polyacrylamide gel without boiling the sample prior to application.After separation, the gel was washed in 50 mM NaHPO₄ buffer, pH 7.0(0.1% Triton X-100) and incubated in the enzymatic substrate ELF 97β-glucuronide diluted in the same buffer (without Triton X-100). Gelswere then fixed overnight in methanol:acetic acid:water (5:1:1). Afterfixing they were washed in dH₂O, equilibrated in 10 mM sodium boratebuffer pH 9.5, and then incubated with 10 μM BODIPY TR-X, SE in the samebuffer. Proteins were viewed using 300 nm UV trans-illumination orepi-illumination. The total protein profile appeared red fluorescentwhile the specific target enzyme was green fluorescent.

27. Total Protein Staining Using BODIPY Succinimidyl Esters.

Membrane staining:Proteins were separated by SDS-polyacrylamide gelelectrophoresis and electroblotted, as described in Example 1. Themembranes were then equilibrated by incubation for 10 minutes (twotimes) in 10 mM sodium borate buffer, pH 9.5. After equilibration, theblots were stained for 30 minutes with 10 μM of any BODIPY succinimidylester in the same buffer. After two 5-10 mL washes in 100% methanol themembranes were rinsed with dH₂O and allowed to dry. Proteins onmembranes were viewed using UV epi-illumination.

Gel staining:Proteins were separated by SDS-polyacrylamide gelelectrophoresis, as described in Example 1. The gels were then fixedovernight in methanol:acetic acid:water (5:1:1). After washing with dH₂O(3×30 minutes), the gels were equilibrated in 10 mM sodium boratebuffer, pH 9.5. Gels were stained by incubating them 10 μM any BODIPYsuccinimidyl ester in the same buffer. Proteins were viewed using UVtrans-illumination.

28. Screening BODIPY Succinimidyl Esters.

Standard protein molecular weight markers were serially diluted two-foldto generate a concentration range of 1000 ng-0.5 ng of protein/lane. Theproteins were separated by SDS-polyacrylamide gel electrophoresis andelectroblotted, as described in Example 1. The membranes wereequilibrated by incubation for 10 minutes (two times) in 10 mM sodiumborate buffer, pH 9.5. After equilibration, two membranes each werestained with one of the BODIPY succinimidyl ester dyes, in Table 2. Theconcentration of dye used was 10 μM in 10 mM sodium borate buffer, pH9.5. Membranes were stained for 30 minutes with agitation. The membraneswere then rinsed two times, 5 minutes each time, in the same buffer. Allmembranes were then washed 3 times, 10 minutes each time, in 100%methanol. After a final rinse in dH₂O, the membranes were imaged with aconstant 3 second exposure on the Bio-Rad Fluor S Max MultiImager orsimilar device. A UV epi-illumination light source and the 520 or 610long pass filters were used. Each dye was then imaged at its optimalexposure time, carefully avoiding signal saturation. Using the QuantityOne software, the trace density for a particular protein band wasdetermined and the background subtracted. The background-subtractedtrace density was plotted versus a range of protein concentration andthe linear dynamic range of detection was determined. Sensitivity wasestablished by determining the lowest amount of protein visible on themembrane. Brightness of the dyes was compared when imaged with a commonexposure time.

TABLE 2 Comparable Brightness Values of 16 BODIPY Succinimidyl Esters(SEs) Intensity @ 3 secs of 250 ng band of protein Substitute BovineSoybean Com- Oval- Serum Carbonic Trypsin pound # Abs Em bumin AlbuminAnhydrase Inhibitor 630/650-X 625 640 169452.2 40215.84 193469.6161528.4 650/665-X 646 660 31163.88 16294.89 30194.71 26319.33 TR-X 588616 436280.4 141786.4 558595.7 533819.3 TMR-X 544 570 382633.5 140534.5564315.7 541307.9 FL Br₂ 530 545 50639.4 30512.31 70927.13 55972.11 R6G528 547 116884 54146.32 227844.1 228890.7 FL C₅ 504 511 36495.0536574.32 63640.23 49177.72 R6G-X 529 547 219786.1 148676.7 386058.2300411.8 FL 502 510 54071.31 65458.51 71486.49 62468.47 530/550 534 551361990.8 226221 438350.6 180694.7 493/503 500 509 24401.56 25658.7654132.56 23722.79 558/568 559 568 130769.9 37031.36 253114.8 254121.8564/570 563 569 383091.9 171803.2 605554.4 563892.1 576/589 575 58878230.08 65072.49 202045.3 101140.1 581/591 581 591 208951.4 160171.6319791.2 144085.5 FL-X 504 510 153667.2 181723.1 282973 188718.2

TABLE 3 Linearity and Sensitivity of Detection Values of 16 BODIPYSuccinimidyl Esters (SEs) and fluorescein isothiocyanate (FITC).Sensitivity (ng of protein) Bovine Soybean Linearity Optimal SerumCarbonic Trypsin Determined Compound # Abs Em Exposure* AlbuminOvalbumin Anhydrase Inhibitor Using CA 630/650-X 625 640 4 secs 7.8 3.91.9-3.9 1.9-3.9  128-fold, R² = .9813 650/665-X 646 660 26 secs 3.9 3.91.9 1.9 256-fold, R² = .9288 16-fold, R² = .9789 TR-X 588 616 4 secs 7.83.9-7.8  3.9 3.9-7.8  128-fold, R² = .981 TMR-X 544 570 3 secs 3.9-7.8 3.9 1.9-3.9 3.9 128-fold, R² = .9552 FL Br₂ 530 545 10 secs 7.8 7.83.9-7.8 7.8-15.6 64-fold, R² = .9786 R6G 528 547 5 secs 7.8  7.8-15.61.9-3.9 15.6  128-fold, R² = .9369 64-fold, R² = .9615 FL C₅ 504 511 10secs 7.8 7.8 3.9-7.8 7.8-15.6 32-fold, R² = .971 R6G-X 529 547 4 secs7.8-15.6 3.9-7.8 3.9 3.9 64-fold, R² = .9623 FL 502 510 15 secs 7.8-15.6 7.8-15.6 7.8 7.8 16-fold, R² = .9872 530/550 534 551 4 secs 7.8-15.615.6  3.9-7.8 7.8-15.6 128-fold, R² = .9577 493/503 500 509 17 secs7.8-15.6  15.6-31.25 3.9-7.8 31.25 128-fold, R² = .9516 558/568 559 5683 secs 7.8-15.6 7.8 3.9 3.9-7.8  512-fold, R² = .9645 564/570 563 569 2secs 3.9-7.8  1.95-3.9  1.95-3.9  7.8 128-fold, R² = .9907 576/589 575588 4 secs 15.6  7.8 3.9-7.8 7.8 128-fold, R² = .9922 581/591 581 591 2secs 7.8-15.6 7.8 3.9 7.8 255-fold, R² = .9657 FL-X 504 510 4 secs3.9-7.8  3.9-7.8 1.9 3.9 256-fold, R² = .9722 FITC 494 519 10-15 sec†15.6-31.25 125    15.6  31.25 64-fold, R² = .9797 32-fold, R² = .9936*Longest exposure without saturation †estimated

As indicated in Table 3, all of the BODIPY dyes tested, which representa wide variety of chemical substituents on the core4-bora-3a,4a-diazaindacene difluoride (dipyrometheneboron difluoride)core structure provided either superior sensitivity or greater linearityor both over FITC for detection of all proteins tested. Furthermore, theBODIPY dyes provided a more uniform sensitivity for protein detectionthat was relatively independent of the protein's structure, a properlynot observed with FITC, which showed considerable differences insensitivity depending on the nature of the protein. BODIPY dyes with theadditional “X” (aminohexanoyl) that absorbed maximally between 495 nmand 640 nm showed particularly high sensitivity, uniformity of stainingand good linear response. Optimal photographic exposure times, which areanother measure of the sensitivity and brightness of the sample weretypically shorter for the BODIPY dyes than for FITC with the preferredBODIPY dyes having an exposure time of 5 seconds or less.

29. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein on Microarrays Using BODIPY FL-X, SE and Wheat GermAgglutinin.

PVDF membrane was precut to a 2.5 cm×7.5 cm size. Membranes were madewet by immersing them in 100% methanol followed by immersion in waterfor 1 minute. Membranes were then placed on 2.5 cm×7.5 cm glass slidesheld to the surface of the glass by surface tension. Four specific,purified proteins including C-1 acid glycoprotein, horseradishperoxidase, immunoglobulin G and soybean trypsin inhibitor were arrayedfrom a source plate (384 well plate) concentration of 0.0625-4 mg/mL inPBS, onto the PVDF strips using a manual slide microarrayer. The manualarrayer was fixed with 4 rows of 8 pins (32 total) with ˜500 microndiameter spot size, 1.125 micron horizontal pitch and 750 micronvertical pitch (pitch=center to center spacing of spots). The proteinswere spotted in replicates of 6 resulting in an array of 192 spotsincluding 24 0 ng control spots. After arraying proteins, the membraneswere allowed to dry. To label total proteins the arrayed membranes wereequilibrated in 35 mL of 10 mM sodium borate buffer, pH 9.5, twice for10 minutes. After equilibration, the membranes were stained for 30minutes with 10 μM BODIPY FL-X, succinimidyl ester in the same buffer.They were then washed twice for 2 minutes in 10 mM sodium borate buffer,pH 9.5, three times for 10 minutes in 100% methanol and finally once for10 minutes in dH₂O. Membranes were then allowed to air dry. All steps,not including equilibration, were performed by placing the membranes in50 mL centrifuge tubes and placing them on a nutator. Specificglycoprotein detection was performed by first washing the membranes for10 minutes, three times in 50 mM Tris, pH 7.5, 150 mM NaCl followed byblocking for 1 hour in the same buffer plus 0.25% MOWIOL 4-88 and 0.2%Tween 20. For detection of glycoproteins, the membranes were incubatedwith 1 μg/mL wheat germ agglutinin-alkaline phosphatase conjugate in abuffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25%MOWIOL 4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detection of theconjugates, the membranes were incubated in 1.25

g/mL DDAO phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH9.5. Proteins were viewed using 300 nm UV epi-illumination. Wheat germagglutinin binds glycoproteins containing N-acetylglucosamine andN-acetylneuraminic acid residues. The total protein profile appearedgreen fluorescent while the targeted glycoproteins (

-1 acid glycoprotein and immunoglobulin G for wheat germ agglutinin)were stained red fluorescent. Arrays can also be imaged using a lasersystem such as the Fuji FLA-3000 imager utilizing the 633 nm excitationfilter and 675 nm emission filter for the DDAO dye and a 473 nmexcitation filter and 520 nm emission filter for the BODIPY FL-X dye.Other BODIPY dyes can be utilized similarly.

30. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein on Microarrays Using BODIPY FL-X, SE and Concanavalin A.

Proteins were arrayed, as described in Example 1. To label totalproteins, the arrayed membranes were equilibrated in 35 mL of 10 mMsodium borate buffer, pH 9.5, twice for 10 minutes. After equilibration,the membranes were stained for 30 minutes with 10 □M BODIPY FL-X,succinimidyl ester in the same buffer. They were washed twice for 2minutes in 10 mM sodium borate buffer, pH 9.5, three times for 10minutes in 100% methanol and finally once for 10 minutes in dH₂O.Membranes were allowed to air dry. All steps, not includingequilibration, were performed by placing the membranes in 50 mLcentrifuge tubes and placing them on a nutator. Specific glycoproteindetection was performed by first washing the membranes for 10 minutes,three times in 50 mM Tris, pH 7.5, 150 mM NaCl followed by blocking for1 hour in the same buffer plus 0.25% MOWIOL 4-88 and 0.2% Tween 20. Fordetection of glycoproteins, the membranes were incubated with 1

g/mL concanavalin A-alkaline phosphatase conjugate in a buffercontaining 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detection of the conjugates, themembranes were incubated with 1.25

g/mL DDAO phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris, pH9.5. Proteins were viewed using 300 nm UV epi-illumination. ConcanavalinA binds to glycoproteins containing

- mannosyl and

-glucopyranosyl residues. The total protein profile appeared greenfluorescent while the targeted glycoproteins (horseradish peroxidase andimmunoglobulin G for concanavalin A) were stained red fluorescent.Arrays can also be imaged using a laser system such as the Fuji FLA-3000imager utilizing the 633 nm excitation filter and 675 nm emission filterfor the DDAO dye and a 473 nm excitation filter and 520 nm emissionfilter for the BODIPY FL-X dye. Other BODIPY dyes can be utilizedsimilarly.

31. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein on Microarrays with BODIPY TR-X, SE and Wheat GermAgglutinin.

Proteins were arrayed as described in Example 1. To label total proteinsthe arrayed membranes were equilibrated in 35 mL of 10 mM sodium boratebuffer, pH 9.5, twice for 10 minutes. After equilibration, the membraneswere stained for 30 minutes in 10

M BODIPY TR-X, succinimidyl ester in the same buffer. They were thenwashed twice for 2 minutes in 10 mM sodium borate buffer, pH 9.5, threetimes for 10 minutes in 100% methanol and finally once for 10 minutes indH₂O. Membranes were allowed to air dry. All steps, not includingequilibration, were performed by placing the membranes in 50 mLcentrifuge tubes and placing them on a nutator. Specific glycoproteindetection was performed by first washing the membranes for 10 minutes,three times in 50 mM Tris, pH 7.5, 150 mM NaCl followed by blocking for1 hour in the same buffer plus 0.25% MOWIOL 4-88 and 0.2% Tween 20. Fordetection of glycoproteins, the membranes were incubated with 1

g/mL wheat germ agglutinin-horseradish peroxidase conjugate in a buffercontaining 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detection of the conjugates, themembranes were incubated with 50

M Amplex Gold in a buffer containing 10 mM Tris, pH 7.5, 1 mM MgCl₂, 1mM ZnCl₂ and 200

M H₂O₂. Proteins were viewed using 300 nm UV epi-illumination. Wheatgerm agglutinin binds glycoproteins containing N-acetylglucosamine andN-acetylneuraminic acid residues. The total protein profile appeared redfluorescent while the targeted glycoproteins (

-1 acid glycoprotein and immunoglobulin G for wheat germ agglutinin)were stained gold fluorescent. Arrays can also be imaged using a lasersystem such as the Fuji FLA-3000 imager utilizing the 633 nm excitationfilter and 675 nm emission filter for the BODIPY TR-X, SE and a 532 nmexcitation filter and 580 nm emission filter for the Amplex Gold. OtherBODIPY dyes can be utilized similarly.

32. Simultaneous Dichromatic Detection of Total Protein and a SpecificTarget Protein on Microarrays with BODIPY TR-X, SE and Concanavalin A.

Proteins were arrayed, as described in Example 1. To label totalproteins, the arrayed membranes were equilibrated in 35 mL of 10 mMsodium borate buffer, pH 9.5, twice for 10 minutes. After equilibration,the membranes were stained for 30 minutes with 10

M BODIPY TR-X, succinimidyl ester in the same buffer. The membranes werewashed twice for 2 minutes in 10 mM sodium borate buffer, pH 9.5, threetimes for 10 minutes in 100% methanol and finally once for 10 minutes indH₂O. Membranes were allowed to air dry. All steps, not includingequilibration, were performed by placing the membranes in 50 mLcentrifuge tubes and placing them on a nutator. Specific glycoproteindetection was performed by first washing the membranes for 10 minutes,three times in 50 mM Tris, pH 7.5, 150 mM NaCl followed by blocking for1 hour in the same buffer plus 0.25% MOWIOL 4-88 and 0.2% Tween 20. Fordetection of glycoproteins, the membranes were incubated with 1

g/mL concanavalin A-alkaline phosphatase conjugate in a buffercontaining 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Tween-20, 0.25% MOWIOL4-88, 0.5 mM MgCl₂, and 1 mM CaCl₂. For detection of the conjugates, themembranes were incubated with 10

g/mL ELF 39 phosphate in a buffer containing 1 mM MgCl₂ and 10 mM Tris,pH 9.5. Proteins were viewed using 300 nm UV epi-illumination.Concanavalin A binds to glycoproteins containing

-mannosyl and

-glucopyranosyl residues. The total protein profile appeared redfluorescent while the targeted glycoproteins (horseradish peroxidase andimmunoglobulin G for concanavalin A) were stained green fluorescent.Other BODIPY dyes can be utilized similarly.

1. A kit for detection of poly(amino acids) immobilized on a solidsurface, said kit comprising: a. a dipyrrometheneboron difluoride dye ofthe formula:

wherein each of R¹ through R⁷ are independently selected from the groupconsisting of H, halogen, -L-Rx, and substituted or unsubstituted C₁-C₆alkyl, aryl, arylethenyl, arylbutadienyl, and heteroaryl; provided thatone or more of R¹ through R⁷ is H, two or more of R¹ through R⁷ isnonhydrogen, and only one of R¹ through R⁷ is -L-Rx, where L is a spacerhaving 1-24 nonhydrogen atoms and Rx is a maleimide or a succinimidylester of a carboxylic acid; such that the dipyrrometheneboron difluoridedye has an absorption maximum between 495 nm and 640 nm; b. a specificbinding pair member that contains a label and that selectively binds toa target that is its complementary binding pair.
 2. The kit according toclaim 1, further comprising a fluorogenic substrate.
 3. The kitaccording to claim 2, wherein the specific binding pair member containsa label that is an enzyme and the enzyme is capable of utilizing thefluorogenic substrate to generate a detectable optical response.
 4. Thekit according to claim 1, wherein the specific binding pair member is anantibody or antibody fragment.
 5. The kit according to claim 1, whereinthe specific binding pair member contains a label that is a fluorescentdye.
 6. The kit according to claim 1, wherein said specific binding pairmember is a biotin-binding protein.
 7. The kit according to claim 6,wherein the biotin-binding protein is avidin, Neutravidin orstreptavidin.
 8. The kit according to claim 1, wherein the label is anenzyme that is a peroxidase or a phosphatase.
 9. The kit according toclaim 8, wherein the peroxidase is horseradish peroxidase.
 10. The kitaccording to claim 2, wherein the fluorogenic substrate is a peroxidasesubstrate that is a fluorescent tyramide.
 11. The kit according to claim8, wherein the phosphatase is alkaline phosphatase.
 12. The kitaccording to claim 2, wherein the fluorogenic substrate is a phosphatasesubstrate that is a 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate.
 13. The kit according to claim 2, wherein the fluorogenicsubstrate is a phosphatase substrate that is a2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone. 14.The kit according to claim 2, wherein the fluorogenic substrate is aphosphatase substrate that is ELF 39 reagent.
 15. The kit according toclaim 1, wherein R¹ is methyl or -L-Rx; R² is H, bromine, or -L-Rx; R³is H or methyl; R⁴ is H or -L-Rx; R⁵ is H, methyl, or phenyl; R⁶ is H orbromine; and R⁷ is methyl, phenyl, alkoxyphenyl, phenylethenyl,phenylbutatdienyl pyrrolyl, or thienyl; where -L- is —(CH₂)₂—, —(CH₂)₄—,—OCH₂C(O)NH(CH₂)₅—, —(CH₂)₂—C(O)NH(CH₂)₅—, —(CH)₂C₆H₄OCH₂C(O)NH(CH₂)₅—;and Rx is a succinimidyl ester of a carboxylic acid; and the specificbinding pair member is an antibody or a streptavidin that contains alabel that is an alkaline phosphatase and the fluorogenic substrate is a9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate, a2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, orELF 39 reagent.